Hepatocellular carcinoma (HCC) is one of the leading causes of mortality worldwide; therefore, searching for an effective treatment for this illness is of great importance. In the present work, in vitro cytotoxic activity of the ethanol extract of the aerial parts of Cynara cardunculus L. against human liver carcinoma cells (Hep G2) was tested. Additionally, the antitumor activity of the extract was confirmed using chemically induced rat liver carcinogenesis with diethylnitrosamine (DEN). Moreover, bioguided fractionation and column chromatographic separation of the active compounds were carried out. The extract of C. cardunculus showed a promising cytotoxic activity according to the protocols of the National Cancer Institute. Bioguided chromatographic separation of the ethanol extract of C. cardunculus led to the isolation of seven secondary metabolites including two sesquiterpene lactones as the principal active components of the methylene chloride soluble fraction, grosheimin (IC50 = 7.49 lg/mL) and cynaropicrin (IC50 = 13.9 lg/mL). The compounds were characterized by different spectroscopic techniques such as EI-MS, IR and NMR. Additionally, in silico analysis of the two active compounds revealed their ability to bind with caspase-3 via hydrogen bonds interactions to initiate apoptosis of cancer cells. The results shed the light on the significance of C. cardunculus as a potential source of antitumor agents.