Cousinia is one of the widespread genera of Asteraceae family. There are numerous studies on this genus and it has not been examined phytochemically in detail. Thus, we aimed to evaluate the phytochemical composition and the cytotoxic effect of Cousinia ermenekensis on human lung cancer (A549), human colon adenocarcinoma (Colo 205), hepatocellular carcinoma (HepG2) cell lines and human bronchial epithelial (Beas-2b) cell line. C. ermenekensis methanol extract showed higher cytotoxic activity against Colo 205 cell line (IC50 = 69 µg/mL). A preliminary examination of the mass spectrums revealed the presence of 13 compounds in the extract. According to the quantitative analyses, the highest content of chlorogenic acid, malic acid, quinic acid and, rutin were detected in extract (28.496, 18.264, 15.115 and 12.506 µg/mg extract respectively).