Scorzonera tomentosa L. (family Asteraceae), endemic to Turkey is reputed for its medicinal attributes in the folkloric medicine. However, only few studies on the chemical composition and pharmacological properties of this plant have been published so far. In this direction, this study aimed to shed light on the phenolic composition, antioxidant and key enzyme inhibitory properties of S. tomentosa aerial parts and roots extracts prepared using different solvents (dichloromethane, ethyl acetate, hexane, water and methanol). Overall, the methanolic extracts and the infusion of both plant parts were better sources of total polyphenolics with 40.33 and 39.30 mg gallic acid equivalent (GAE)/g, respectively, while for the aerial parts, 22.42 and 21.84 mg GAE/g were measured for methanolic extract and root infusion, respectively. Extracts of the aerial parts showed considerable amount of total flavonoid content (2.96–34.93 mg rutin equivalent (RE)/g) as compared with the root extract (0.40–2.65 mg RE/g), with highest content observed in methanolic extract. For this reason, the methanolic extract of roots and aerial parts were subjected to liquid chromatography coupled to diode array detection and electrospray ionization tandem mass spectrometry (LC-DAD-MSn) analysis. The methanolic and aqueous extracts of both plant parts proved to be most significant radical quenchers (1.05–45.89 mg trolox equivalent (TE)/g in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay; and 1.82–82.94 mg TE/g in 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay) and reducing agents (39.40–164.06 mg TE/g in cupric reducing antioxidant capacity (CUPRAC) and 15.54–79.78 mg TE/g in ferric reducing antioxidant power (FRAP)). The dichloromethane extracts were significant metal chelators (13.92 and 6.24 mg ethylenediaminetetraacetate equivalent (EDTAE)/g for aerial parts and roots). Extracts of both plant parts showed selective inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Notable anti-tyrosinase activity was observed for all the extracts (aerial part: 16.40 ± 1.30 - 64.83 ± 0.28 and roots: 6.29 ± 0.50–65.46 ± 0.31 mg kojic acid equivalent (KAE)/g), while modest inhibitory activity against α-amylase (0.08−0.61 mmol acarbose equivalent (ACAE)/g) and α-glucosidase (0.09−0.83 mmol ACAE/g) for all the tested extracts. In addition, to provide the first scientific insight on the biological and chemical profile of S. tomentosa, observations from this present study tend to identify this plant as a valuable source of biologically active molecules for possible phytopharmaceutical and cosmeceuticals exploitation.