Bacterial and fungal infections, along with the ability of microorganisms to form biofilms, remain significant health concerns that may increase infection persistence and reduce the effectiveness of antimicrobial therapy. The development of natural-product-based antimicrobial agents with both antimicrobial and antibiofilm activities represents a promising approach. Songga wood (Strychnos ligustrina) is a potential source of bioactive compounds with antimicrobial properties. This study aimed to formulate ethanol extract of songga wood into a thermoresponsive gel spray and evaluate its antibacterial, antifungal, and antibiofilm activities against pathogenic microorganisms. The ethanol extract of songga wood was formulated into three preparations based on extract concentration: F1 (1%), F2 (2%), and F3 (4%). Antibacterial activity was evaluated against Staphylococcus aureus and Salmonella typhi, while antifungal activity was assessed against Candida albicans and Candida tropicalis using the microdilution method. Chloramphenicol was used as the positive control for the antibacterial assay, and nystatin was used as the positive control for the antifungal assay. Antibiofilm activity was evaluated using the crystal violet assay after 24 and 48 hours of incubation. The results showed that increasing the extract concentration enhanced the inhibitory activity, with F3 (4%) exhibiting the highest activity. F3 demonstrated antibacterial inhibition of 97.1% against S. typhi and 95.6% against S. aureus, while antifungal inhibition reached 96.7% against C. albicans and 96.5% against C. tropicalis. These activities indicated strong inhibitory effects that were comparable to those of the positive controls. In the antibiofilm assay, F3 showed inhibition of S. typhi by 91.2% and 78.5%, S. aureus by 82.9% and 79.1%, C. albicans by 87.2% and 80.5%, and C. tropicalis by 91.2% and 86.9% after 24 and 48 hours, respectively. Overall, the antibiofilm activity of F3 was higher at 24 hours than at 48 hours, although strong inhibitory activity was maintained. Conclusion: The thermoresponsive gel spray containing ethanol extract of songga wood exhibited strong antibacterial, antifungal, and antibiofilm activities, with Formula 3 (4%) demonstrating the greatest potential. These findings highlight S. ligustrina as a promising natural-product candidate for the development of antimicrobial and antibiofilm preparations.