Objective: To find the cytotoxic and apoptotic effects of neem oil extract on the selected cancerous (A-549, PC-3 and DU-145) and noncancerous (NIH3T3 and CCD-18Co) cell lines. Methods: Viability and cytotoxic effect induced by the extract was measured by using MTT assay and apoptotic effect of the extract was evaluated by using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and activity of caspases 3, 8 and 9 through colorimetric assay kits. Results: The results showed that neem oil extract significantly reduced the viability in all selected cancer cells treated with varying concentrations of extract as compared with untreated cells and had less effect on noncancerous cell lines. It significantly increased the percentage of necrotic and apoptotic cells, and caspases 3, 8 and 9 activities in all cancer cells treated with extract as compared with untreated cells whereas no effect on noncancerous cell lines. It suggested that neem oil extract exerted a higher cytotoxic effect on cancer cells than normal cells and lower concentration induced apoptosis only in cancer cells. One of the apoptosis-inducing mechanism was through the activation of caspases signaling pathways. Conclusion: Conclusively, it implies that neem oil extract may contain one or more potential agents that can be used as a safe and effective anticancer therapy.