Medicinal herbs are significant sources of chemotherapeutic drugs and play a vital role in the prevention and treatment of cancer. Mentha pulegium L. from Labiatae family was traditionally used as an anticancer agent. In this study, aerial parts including leaves of this plant were extracted by methanol and fractionated extracts have been produced by petroleum ether, ethyl acetate, acetone, methanol and distilled water. For the purpose of cytotoxic evaluation of methanolic extract and its fractions on human ovary carcinoma cells (C13), human hepatocarcinoma cells (HepG2) and human lung carcinoma cells (A549), clonogenic assay was performed. Briefly, 200 cells were seeded in each well of 6 well plates in RPMI 1640 with 10% FBS media. After 24 hours incubation, 0-50μg/ml of methanolic extract and its fractions were exposed to the cells. Finally, colonies with more than 50 cells were counted after 7 days. In each case, a control row was set by the exposure of cells to compounds-free solvents. LC50 values were calculated using nonlinear regression analysis on Graphpad prism® software. The result showed that the methanolic extract and its fractions are cytotoxic on all three studied human carcinoma cell lines at different degrees. Human ovary carcinoma cell line (C13), which is resistant to many other chemotherapeutic agents (e.g. cisplatin), is the most sensitive cell line to methanolic extract and its fractions compared to two other cell lines. Further complementary cellular and animal studies are recommended for these anticancer candidates.