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Despite newly introduced treatment options, the 5-year survival rate for non-small cell lung cancer (NSCLC) remains below 20%, indicating a remaining need for new treatment strategies. Benzamil (BZ) has demonstrated inhibitory effects on brain tumors and osteosarcoma; however, its effects on NSCLC cells remain unknown. In this study, we investigated the potential anti-cancer effects of BZ in a panel of NSCLC cell lines. Compared to cells with EGFR L858R/T790M double mutations (H1975), BZ induced greater cytotoxicity in EGFR wild-type (A549, H1299, H292) and EGFR ex19del (PC9) cells. Mechanistically, BZ treatment was associated with inhibition of Akt, ERK1/2, and Stat3 signaling and increased mitochondrial damage. It was also associated with reduced Drp1 and Fis1 expression. Moreover, BZ induced release of cytochrome c, Smac/Diablo, and HtrA2/Omi from mitochondria. Additionally, BZ enhanced osimertinib-induced cytotoxicity in EGFR-mutant cells, and it suppressed H1975-derived cancer spheres. These findings support a proposed model in which BZ-induced mitochondrial damage is accompanied by the release of cytochrome c, Smac/Diablo, and HtrA2/Omi. These changes were accompanied by caspase activation and cell line-dependent alterations in XIAP expression, consistent with their potential contribution to apoptosis in NSCLC cells.