Members of the Onosma genus are widely used in folk medicine and they have great interest in the pharmaceutical industry. In this study, phytochemical characterization, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), metal chelation, and phosphomolybdenum assays), enzyme inhibition (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-amylase, α-glucosidase, and tyrosinase), antimicrobial activity (microdilution method), genotoxic and antigenotoxic (by using Drosophila melanogaster larvae and Comet assay) potentials of Onosma sericea Willd. and Onosma stenoloba Hausskn. ex Riedl. were investigated. Additionally, the bioactive compounds were identified by high performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) analysis. Generally, O. sericea showed stronger antioxidant activity, while O. stenoloba extract exhibited stronger enzyme inhibitory abilities (cholinesterases and α-amylase). The protective effects of extracts, at the concentration range from 25 to 400 μg/mL, against hydroxyl radical-induced DNA damage were dose-dependent, increasing with a higher dosage. The extracts at the highest concentration (80 mg/mL) showed the absence of genotoxicity in vivo. Antigenotoxic effects were evident after treatment with both extracts, with a percentage reduction of over 80 %. Overall antimicrobial activity of studied extracts was weak, with the lowest minimal inhibitory concentration values (MIC) of 2.5 mg/mL. Taken together, obtained results showed that tested Onosma species can be considered as promising sources of bioactive phytochemicals for pharmacological purposes.