BACKGROUND: Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the leading causes of cancer-related deaths worldwide. Vincristine (VCR) is a chemotherapeutic agent for lung cancers; however, its effectiveness is limited by side effects and the development of drug resistance. Patchouli alcohol (PA), from Pogostemon cablin extract, is known to possess anti-inflammatory and anticancer properties. In this study, we investigated the role of PA in inducing reactive oxygen species (ROS)-mediated DNA damage in A549 and VCR-resistant A549/V16 NSCLC cells. METHODS: The anticancer potential of PA was studied using the MTT assay, colony formation, flow cytometry analysis, western blotting, DCFDA staining, immunofluorescence staining, and TUNEL assay techniques. RESULTS: The intracellular ROS levels were enhanced in PA-treated cells, activating the CHK1 and CHK2 signaling pathways. PA further inhibited proliferation and colony-forming abilities and induced cell cycle arrest at the G0 /G1 phase by regulating p53/p21 and CDK2/cyclin E1 expression. Moreover, PA increased the percentage of cells in the subG1 phase and induced apoptosis by activating the Bax/caspase-9/caspase-3 intrinsic pathway. In addition, drug resistance (p-glycoprotein) and cancer stem cell (CD44 and CD133) markers were downregulated after PA treatment. Furthermore, combining PA and cisplatin exhibited synergistic inhibitory activity in A549 and A549/V16 cells. CONCLUSIONS: PA induced ROS-mediated DNA damage, triggered cell cycle arrest and apoptosis, attenuated drug resistance and cancer stem cell phenotypes, and synergistically inhibited proliferation in combination with cisplatin. These findings suggest that PA has the potential to be used for the treatment of NSCLC with or without VCR resistance.