Phytochemical composition of ethyl acetate fraction and total phenolic content, in vitro antioxidant, anti-inflammatory, antimicrobial activities of petroleum ether, chloroform, ethyl acetate and n-butanol fractions of the ethanol extract obtained from the subaerial parts of Scorzonera pygmaea Sibth. & Sm. (Asteraceae) were investigated. Nine compounds; scorzopygmaecoside (1), scorzonerol (2), cudrabibenzyl A (3), thunberginol C (4), scorzocreticoside I (5) and II (6), chlorogenic acid (7), chlorogenic acid methyl ester (8), 3,5-di-O-caffeoylquinic acid (9) were isolated and identified using spectroscopic methods. All substances were isolated for the first time from this species. Compounds 1 and 2 are new. The fractions showed high antioxidant capacity correlated with their phenolic content and no significant antimicrobial activity against tested bacteria and fungi. COX inhibition test was used to evaluate the anti-inflammatory activity and all the fractions showed low inhibition in comparison with indomethacin.