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• Phytochemistry and biological activity of Onosma sieheana and O. stenoloba were studied. • Onosma sieheana exhibited higher antioxidant and α-amylase inhibitory activity than the other. • Tyrosinase inhibitory activity test resulted in the superiority of O. stenoloba. • The binding energies of most of the main components to enzymes was quite high. There are convincing evidences that species belonging to the Onosma genus have been used in the treatment of various diseases for centuries. In this study, antioxidant and tyrosinase and α-amylase inhibitory activities of the methanol (MeOH) extracts of two endemic Onosma species (O. sieheana Hayek and O. stenoloba Hausskn. ex Riedl), which are endemic to the flora of Turkey, were compared. Phosphomolybdenum, reducing power [Cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP)], radical scavenging [on '2,2-diphenyl-1-picrylhydrazyl' (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS)] and ferrous ion chelating assays were used to determine the antioxidant activities of the extracts. All test systems except for the ferrous ion chelating assay resulted in O. sieheana 's superiority. The antioxidant activity of O. sieheana in the test systems mentioned above was found to be 2.11, 1.57, 1.01, 3.54 and 3.19 mg/mL, respectively. However, in ferrous ion chelating activity assay, O. stenoloba showed higher activity with an IC 50 value of 2.98 mg/mL. In the tyrosinase inhibitor activity test, O. stenoloba extract showed higher activity (1.89 mg/mL), while in the α-amylase inhibitory activity assay, O. sieheana was found to be more effective (3.02 mg/mL). As a result of total phenolic and flavonoid compound analysis, O. sieheana was found to be richer than the other in terms of both groups. LC–ESI–MS/MS analysis revealed that rosmarinic acid, apigenin 7-glucoside, luteolin 7-glucoside, chlorogenic acid, hesperidin, pinoresinol, hyperoside and 4-hydroxybenzoic are the main compounds of the extracts. According to the results of docking analysis performed to reveal the interactions of the main components with tyrosinase, it is thought that pinoresinol, hesperidin, luteolin 7-glucoside, apigenin 7-glucoside, and hyperoside may be phytochemicals responsible for the inhibitory activity. Additionally, the binding energies of pinorecinol, apigenin 7-glucoside, luteolin 7-glucoside, hyperoside, chlorogenic acid, and rosmarinic acid to α-amylase were found to be quite high. [ABSTRACT FROM AUTHOR]