The paper deals with the isolation of the constituents of Xanthium spinosum L. and the determination of its biological activity (toxicity). The taxon which is found also in our flora has not been studied from this aspect yet. The used methodology is of a bioassay-guided separation character. The Tubifex tubifex test (TT test) served as the basic biological test. From the methanol extract of the aerial part, ent-kaurane diterpene atractyloside was isolated, from the acetone extract of the aerial part, xanthumin and deacetylxanthumin, two derivatives of xanthanolides – sesquiterpene lactones, were isolated, and from the ethanol extract, phytosterols – β-sitosterol and stigmasterol. From the acetone extract of fruits, fatty acids were obtained.

        Key words: Xanthium spinosum L. – Asteraceae – bioassay-guided separation – atractyloside – xanthanolides – Tubifex tubifex test