In the current study, the n-hexane, chloroform, ethyl acetate, methanol, and water extracts of Phagnalon graecum Boiss. (Asteraceae), a spice plant, were investigated for their enzyme inhibitory activity against acetylcholinesterase, butyrylcholinesterase, lipoxygenase, tyrosinase, and antioxidant activities. Antioxidant activity of the extracts was determined by 2,2-diphenyl-1-picrylhydrazyl radical scavenging, ferric ion-chelation activity, and ferric-reducing antioxidant power tests. Total phenol (gallic acid equivalent) and flavonoid (quercetin equivalent) contents were calculated. Essential oil of the plant was analyzed by GC-MS, while the selected phenolic acids were analyzed by HPLC. The extracts had insignificant inhibition against the tested enzymes, whereas they displayed a remarkable antioxidant activity. Therefore, to the best of our knowledge, this study presents the first data on biological activity and phytochemical content of P. graecum and this species could be utilized as potential antioxidant applicable for food preservation.