Diabetic ulcers are difficult to treat due to impaired healing and microbial biofilms that evade immune responses and reduce antimicrobial sensitivity. This study investigates the phytochemical profile and antibacterial and antibiofilm activities of Etlingera balikpapanensis methanol extract against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The extract was formulated into chitosan-coated poly(ε-caprolactone) nanoparticles within a thermosensitive gel, then characterized and evaluated for accelerating infected diabetic wound healing. E. balikpapanensis rhizomes obtained from Batu Timbau Village, East Kutai, East Kalimantan, were botanically authenticated, dried at 40 °C, ground, and macerated using methanol at a solid-to-solvent ratio of 1:10 (w/v) for 72 hours. Extraction was performed in three groups to evaluate the consistency of the process. The extraction yields for each group were 10.02%, 10.15%, and 10.25%, with an average yield of 10.14 ± 0.12% and a total crude extract mass of 25.36 g from 250 g of dried rhizome powder. Low intergroup variability indicates that the extraction procedure yields relatively consistent results. Qualitative phytochemical screening using specific reactions revealed the presence of alkaloids, flavonoids, saponins, and terpenoids. The presence of these metabolite groups provides a chemical basis for the biological activity of the extracts, as alkaloids, flavonoids, saponins, and terpenoids are known to affect bacterial membrane integrity, cell adhesion, intercellular communication, extracellular matrix production, and the expression of virulence factors. The chemical profile of the extract was characterized using high-resolution liquid chromatography–tandem mass spectrometry in positive ion mode. The analysis yielded a large number of molecular features during the 25-minute chromatographic separation. Based on accurate mass, isotope patterns, tandem fragmentation, and matching against the mzCloud spectral library, several metabolites were putatively annotated, including choline, betaine, methyl isonicotinate, nicotinamide, L-pyroglutamic acid, 5-hydroxymethyl-2-furaldehyde, chlorogenic acid, isovanillic acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, oxidized fatty acid derivatives, methyl octadecatrienoate, and linoleic acid. A retention time feature at approximately 5.19 minutes showed a precursor ion at m/z 165.0544 [M+H]⁺, a dehydrated ion at m/z 147.0437 [M+H−H₂O]⁺, as well as product ions at m/z 147.0439, 119.0492, and 91.0544, which support its putative annotation as p-coumaric acid. However, since the study did not use authentic standards analyzed under identical conditions, all of these determinations should be considered tentative annotations rather than definitive identifications. Signals associated with N,N-diisopropylethylamine and bis(2-ethylhexyl) phthalate are also treated as possible exogenous contaminants until they can be confirmed through solvent blank and procedural blank analyses. The antibacterial activity of the extract was tested using the microdilution method at concentrations of 0.125–1% (w/v). The extract inhibited the growth of all three bacteria at all tested concentrations and showed a trend of increasing inhibition as the concentration increased. At the lowest concentration of 0.125%, the inhibition percentages against S. aureus, P. aeruginosa, and E. coli were 60.10%, 60.12%, and 68.32%, respectively. At a concentration of 1%, these values increased to 78.10%, 75.00%, and 85.90%. E. coli consistently exhibited the highest sensitivity, while P. aeruginosa tended to show the lowest response. Inhibition by the 1% extract was still numerically lower than that of chloramphenicol, which produced inhibition of 82.33%, 81.06%, and 87.11% against these three bacteria. Nevertheless, the crude extract’s ability to produce inhibition ranging from 75.00% to 85.90% indicates substantial antibacterial activity and the possible collective or synergistic contribution of various metabolites within the extract. Antibiofilm activity was assessed using the crystal violet method during the biofilm formation phase at 24 and 48 hours, as well as on established biofilms. At a concentration of 1%, the extract inhibited 24-hour biofilm formation by 81.10% in S. aureus, 79.90% in P. aeruginosa, and 80.30% in E. coli. In 48-hour-old biofilms, the inhibition rates were 78.20%, 75.10%, and 77.50%, respectively. Treatment of established biofilms reduced biomass by 70.10%, 72.30%, and 76.20%. The lower inhibition rate in mature biofilms compared to developing biofilms suggests that established biofilm structures are more difficult to eliminate. This is likely related to the formation of an extracellular polymeric matrix, physiological heterogeneity of the population, decreased metabolic activity, and the presence of persistent cell subpopulations. Although the extract did not completely eliminate mature biofilms, its ability to reduce biomass by more than 70% demonstrates significant potential for the development of adjunctive therapies for infected chronic wounds. These results were supported by observations using SEM analysis. The untreated biofilm exhibited dense cell aggregates, extensive surface coverage, and extracellular matrix-like material that formed a compact and continuous biofilm structure. In contrast, biofilms treated with the extract exhibited fewer clusters of adherent cells, a more dispersed cell distribution, a larger open surface area, and a matrix that appeared less continuous. As an exploratory analysis related to diabetes, the putatively annotated p-coumaric acid was tested for molecular binding to the GH13_30 CmmB α-glucosidase. p-Coumaric acid obtained a binding score of −4.75 kcal/mol and was predicted to interact with residues ASP A:221, ARG A:428, and ARG A:364. These values are substantially less favorable compared to the co-crystallized ligand akarviosin with a score of −9.80 kcal/mol and the comparator inhibitor miglitol with a score of −8.28 kcal/mol. Although the ARG A:428 residue is also involved in the interactions of the comparison ligands, p-coumaric acid occupies a more restricted portion of the binding pocket and does not reproduce several key interactions of akarviosin and miglitol. Therefore, the binding results only indicate the potential for occupancy in specific binding regions and do not demonstrate α-glucosidase inhibitory activity. To improve dispersion and topical delivery, the 1% extract was incorporated into poly(ε-caprolactone) nanoparticles developed using the solvent evaporation-emulsification method and chitosan coating. Based on particle size, polydispersity index, and surface charge, the NPs1 formulation was selected as the best candidate. This formulation had a mean hydrodynamic diameter of 248.6 ± 6.21 nm, a polydispersity index of 0.196 ± 0.008, and a zeta potential of +34.8 ± 1.42 mV. The relatively low polydispersity index indicates a fairly uniform size distribution, while the positive charge is consistent with the presence of chitosan on the nanoparticle surface and supports the electrostatic stabilization of the system. The selected nanoparticles were then dispersed into gels based on poloxamer 407, poloxamer 188, and hydroxypropyl methylcellulose K100M. Of the six screened gel compositions, the NPs–Tgel 1 formulation was selected because it underwent a sol–gel transition at a temperature close to physiological temperature. The final formulation was a slightly cloudy, odorless, homogeneous liquid that showed no visual signs of aggregation or phase separation during the 28-day observation period. The gel had a pH of 6.41 ± 0.05, a sol–gel transition temperature of 36.5 ± 0.3 °C, an expansion ratio of 1.48 ± 0.05, a spreadability of 6.2 ± 0.3 cm, and an adhesiveness of 13.1 ± 0.6 seconds. Viscosity increased significantly from 291.3 cP at 4 °C to 689.3 cP at 25 °C and 28,145.1 cP at 37 °C. These properties indicate that the formulation remains relatively easy to apply at low or room temperatures but forms a much thicker system upon reaching body temperature. This increase in viscosity has the potential to prolong the formulation’s contact time with the wound surface and reduce runoff or loss of the preparation after application. Wound-healing potential was evaluated using male Wistar rats with alloxan-induced diabetes. Full-thickness excision wounds with a diameter of 10 mm were created on the dorsal region and inoculated with S. aureus ATCC 6538. The animals were grouped into the following: infected diabetic wound control (no treatment), empty gel control, chloramphenicol positive control, non-diabetic wound control, and the thermosensitive gel group loaded with nanoparticle extracts. The formulations were administered topically once daily for 14 days. The untreated group showed the slowest healing with a wound contraction of 61.70 ± 2.60% on day 14, whereas the empty gel group achieved a contraction of 78.90 ± 2.00%. The findings in the empty gel group suggest that the gel base itself provides benefits, likely through the creation of a moist and protected wound environment. The thermosensitive gel loaded with nanoparticles of the extract produced wound contraction of 23.70 ± 2.20% on day 3, 68.70 ± 2.50% on day 7, and 98.20 ± 0.90% on day 14. At the end of the observation period, wound contraction in the active formulation group was significantly greater than in the untreated group and the placebo gel group, with a corrected p-value <0.001 for both comparisons. The response of the active formulation did not differ significantly from that of chloramphenicol, which produced a contraction of 95.80 ± 1.20% (corrected p = 0.127), nor from the non-diabetic wound control, which reached 97.60 ± 1.00% (corrected p = 0.970). These results indicate that the combination of the extract, nanoparticle carrier, and thermosensitive gel is associated with accelerated healing of infected diabetic wounds. Overall, E. balikpapanensis rhizome extract showed broad-spectrum antibacterial and antibiofilm activities. Its nanoparticle-based thermosensitive gel had suitable physicochemical properties and accelerated infected diabetic wound healing, with day-14 outcomes comparable to chloramphenicol and the non-diabetic control. These findings support its potential as an antibiofilm agent for topical wound treatment.