The genus Onosma is the biggest genus in the family Boraginaceae and its members are widely used for traditional purposes in several countries folk medicine. In the current work, two Onosma (O. trapezuntea and O. rigidum) species were examined for biological properties and chemical characterization. To evaluate biological properties, antioxidant and enzyme inhibitory assays were performed. Antioxidant assays included free radical scavenging (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)), reducing power (cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP)), metal chelating and phosphomolybdenum. Enzyme inhibitory were assessed against cholinesterase, tyrosinase, α-amylase and α-glucosidase. Total phenolic and flavonoid contents in O. trapezuntea were higher than those of O. rigidum. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that chlorogenic acid, rosmarinic acid and hyperoside were dominant compounds in the chemical profiles of the tested Onosma extracts. Generally, O. trapezuntea exhibited stronger antioxidant abilities than O. rigidum. However, the best chelating ability was recorded in O. rigidum. In addition, O. rigidum was more active on cholinesterase and α-glucosidase when compared with O. trapezuntea. The tested extracts exhibited similar tyrosinase inhibitory potentials. Consequently, our findings suggested that the tested Onosma species could be presented as valuable sources of natural ingredients such as antioxidant and enzyme inhibitors.