Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality, with therapeutic resistance posing a critical obstacle in treatment. This study examines the anticancer effects of deoxybouvardin (DB), a cyclic hexapeptide, in both gefitinib (GEF)-sensitive and -resistant HCC827 NSCLC cells. DB exhibited a dose- and time-dependent reduction in cell viability and proliferation. In vitro kinase assays demonstrated that DB effectively inhibited epidermal growth factor receptor (EGFR), mesenchymal–epithelial transition (MET), and AKT, leading to decreased phosphorylation of these key signaling molecules. Molecular docking analysis further suggested that DB interacts with the ATP-binding sites of these kinases. Additionally, DB treatment elevated reactive oxygen species (ROS) levels, induced cell cycle arrest, and disrupted mitochondrial membrane potential DB-induced apoptosis was caspase-dependent, as shown by its inhibition upon treatment with Z-VAD-FMK, a pan-caspase inhibitor. Overall, DB exerted significant anticancer effects in both GEF-sensitive and -resistant NSCLC cells by concurrently targeting EGFR, MET, and AKT, while also enhancing ROS accumulation and promoting caspase-dependent apoptosis. These findings suggest that DB holds promise as a potential therapeutic approach for overcoming drug resistance in NSCLC.