Introduction: Cancer continues to be a prevalent health problem with increasing mortality rates. The interest in complementary medicine for cancer treatment has increased in recent years. In this study, we aimed to investigate whether there are in vitro anticancer effects of a commercially available polyherbal formulation (PHF) against lung cancer. Methods: MTS assay, colony formation, wound healing, Annexin-V/PI assays and qRT-PCR were performed to determine the effects of PHF on viability, colony forming ability, migration and apoptosis of non-small cell lung cancer (NSCLC) cells, A549 and human retinal epithelial cells, ARPE-19 as a non-cancerous control. Results: We examined the cytotoxic effect of PHF on cell proliferation in comparison with cisplatin and both showed significant cytotoxic effects on A549 cells. PHF treatment resulted in a relatively mild cytotoxic effect against ARPE-19 cells compared to cisplatin treatment. The inhibition of growth in more than 50% of A549 cells was noted with either 1:25 dilution of PHF or 24 mu M of cisplatin treatments. PHF caused a more effective suppression of cell migration compared to cisplatin (P<0.001). Both PHF and cisplatin at IC50 doses effectively inhibited colony formation and induced apoptosis. Bax/Bcl-2 ratio was elevated in A549 cells after both PHF and cisplatin treatments. Conclusion: Our data indicate that PHF might have potent migration inhibitory and apoptosis-inducing activities. More importantly, PHF showed a less cytotoxic effect on normal cells than cisplatin. The mechanisms underlying the anticancer activity of PHF need to be elucidated more comprehensively to confirm its potential as a promising therapeutic candidate.