Onopordum acanthium has been considered in traditional medicine to be effective against different cancers. The chloroform extracts of the root, which displayed antiproliferative effect against human tumor cell lines, was subjected to bioactivity-guided multistep chromatographic separation. This experiment resulted in the isolation of the sesquiterpene lactones 4beta,14-dihydro-3-dehydrozaluzanin C (1), zaluzanin C (2) and 4beta,15,11beta,13-tetrahydrozaluzanin C (3), the neolignan nitidanin-diisovalerianate (4), besides 13-oxo-9Z,11 E-octadecadienoic acid (5), 24-methylenecholesterol (6), alpha-linolenic acid, linoleic acid, stigmasterol and beta-sitosterol. The structures of the isolated compounds were established through analytical data (NMR, MS), and by comparison of these with those reported in the literature. All the aforementioned compounds were detected for the first time from this plant. The antiproliferative activities of compounds 1-6 were assessed on cervix adenocarcinoma HeLa, breast adenocarcinoma MCF7 and skin epidermoid carcinoma A431 cells by using the MTT assay. It was found that, 4beta,14-dihydro-3-dehydrozaluzanin C (1), the most active antiproliferative compound of the extract, exerted remarkable tumor cell growth inhibitory activity (IC50 2.7-15.1 microM).