Non-small cell lung cancer (NSCLC) remains a major cause of cancer mortality, with resistance to targeted therapies limiting treatment efficacy. Epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) are key therapeutic targets in NSCLC. This study investigates the anticancer effects of deoxybouvardin glucoside (DBG), a natural compound isolated from Rubia philippinensis, and its molecular mechanisms in NSCLC cells. DBG significantly inhibited the proliferation of HCC827 and gefitinib-resistant HCC827GR cells in a dose-dependent manner, demonstrating potent anticancer activity even at nanomolar (nM) concentrations. In vitro kinase assays and molecular docking confirmed DBG as a direct inhibitor of EGFR, MET, and AKT at nM potency, effectively suppressing tumor-promoting signaling. Western blot analysis further showed reduced phosphorylation of these kinases, leading to downstream inhibition. Flow cytometry revealed that DBG induced G0/G1 phase arrest, elevated reactive oxygen species (ROS) levels, and activated caspase-dependent apoptosis. NAC pretreatment mitigated ROS generation and apoptosis, confirming oxidative stress involvement in DBG’s mechanism. These findings suggest that DBG exerts strong anticancer effects by directly targeting the EGFR/MET/AKT pathway at nM concentrations, even in drug-resistant NSCLC cells. This underscores its potential as a promising therapeutic candidate for overcoming resistance in NSCLC.