Scorzonera L. (Asteraceae) species are widespread and utilised as traditional medicine and edible plant. A phytochemical study on ethyl acetate fraction obtained from the ethanol extract of Scorzonera hieraciifolia Hayek (an endemic species of Turkey) roots resulted in isolation of nine compounds; 5-O-feruloyl quinic acid methyl ester (1), 1,5-di-O-feruloylquinic acid (2), chlorogenic acid methyl ester (3), 3-O-caffeoylquinic acid methyl ester (4), 1,3-di-O-caffeoylquinic acid methyl ester (5), 3,5-di-O-caffeoylquinic acid methyl ester (6), 4,5-di-O-caffeoylquinic acid methyl ester (7), caffeic acid (8), 3-(4’-Hydroxyphenyl)-2-propenoic acid (4″-carboxyl)-phenyl ester (9). Structures of the compounds were determined using spectral methods. All substances were isolated for the first time from this species and compound 9 is new for the family Asteraceae. In vitro antioxidant, anti-inflammatory and antimicrobial activities of petroleum ether, chloroform, ethyl acetate and n-butanol fractions were evaluated. Ethyl acetate fraction showed the highest amount of total phenolic content and antioxidant capacity. No significant antimicrobial activity was determined against tested bacteria and fungi. COX inhibition test was employed for anti-inflammatory studies, all fractions showed low inhibitory effect on both COX-1 and COX-2 enzymes in comparison with indomethacin.