Drug resistance is a major challenge in cancer treatment. The substrates of NAD(P)H:quinone oxidoreductase 1 (NQO1) show a promising anticancer effect in clinical trials. We previously identified a natural NQO1 substrate 2-methoxy-6-acetyl-7-methyljuglone (MAM) with a potent anticancer effect. The present study was designed to explore the efficacy of MAM in fighting against drug-resistant non-small cell lung cancer (NSCLC). The anticancer effect of MAM was evaluated in cisplatin-resistant A549 and AZD9291-resistant H1975 cells. The interaction of MAM with NQO1 was measured by cellular thermal shift assay and drug affinity responsive target stability assay. The NQO1 activity and expression were measured using NQO1 recombinant protein, Western blotting, and immunofluorescence staining assay. The roles of NQO1 were examined by NQO1 inhibitor, small interfering RNA (siRNA), and short hairpin RNA (shRNA). The roles of reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation were determined. MAM induced significant cell death in drug-resistant cells with similar potency to that of parental cells, which were completely abolished by NQO1 inhibitor, NQO1 siRNA, and iron chelators. MAM activates and binds to NQO1, which triggers ROS generation, LIP increase, and lipid peroxidation. MAM significantly suppressed tumor growth in the tumor xenograft zebrafish model. These results showed that MAM induced ferroptosis by targeting NQO1 in drug-resistant NSCLC cells. Our findings provided a novel therapeutic strategy for fighting against drug resistance by induction of NQO1-mediated ferroptosis.