Context The genus Anthemis L. (Asteraceae) comprises about 195 species which are widely used in the pharmaceutical, cosmetic and food industries. Objective Anthemis mirheydari Iranshar, an endemic plant from Iran, was investigated for its cytotoxic properties and chemical constituents. Materials and methods The whole parts of the plant (320 g) were extracted by dichloromethane and methanol for four days, successively. The cytotoxic activity of both dichloromethane and methanol extracts were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric methods against three human cancer cell lines including LS180, MCF-7 and MOLT-4. Different concentrations (10-100 mg/mL) of the plant extracts were tested to obtain IC 50 values. The dichloromethane extract of A. mirheydari was subjected to silica gel-column and thin layer chromatography for purification of its chemical constituents and the isolated compounds were further tested against MOLT-4 cells. The structures of the pure compounds were elucidated using different spectral data including nuclear magnetic resonance and electron impact mass spectra. Results The IC 50 values of the dichloromethane extract were 30.8 ± 6.7, 25.2 ± 6.5 and 8.6 ± 1.1 mg/mL (means ± standard error) for the above-mentioned cell lines, respectively. Two triterpenoids, taraxasterol (1) and pseudotaraxasterol (2), one sterol, b-sitosterol (3) and one coumarin, 7-methoxycoumarin (4) were isolated from the extract. The IC 50 of the mixture of compounds 1 and 2 as well as compounds 3 and 4 were higher (4100 mM) than that reported for the dichloromethane extract against MOLT-4 cells. Conclusion The dichloromethane extract was the most active one among the tested material.ARTICLE HISTORY