Introduction The present study was designed to evaluate the phytoconstituents, antioxidant, enzyme inhibitory, antimicrobial, and cytotoxic properties of the aerial parts and root extracts of A. amoenus, A. microcephalus , A. sterocalyx, A. tigridis , and A. yuksellii . Methods The chemical composition was evaluated by spectrophotometric methods and LC–MS-qTOF metabolomic analysis. Antioxidant properties were examined by in vitro chemical assays. The enzyme inhibitory effects were investigated using key enzymes. Antibacterial and antifungal effects were observed against various bacterial and fungal strains. DU-145 (prostate cancer), HeLa (cervical adenocarcinoma), A549 (lung adenocarcinoma), and HEK-293 (cells derived from embryonic kidney tissue) cell lines were used for evaluating cytotoxic effects. Results Metabolomic analysis revealed that hydroxybenzoic acid, hydroxycinnamic acid, and methyl glutamic acid were key metabolites in the aerial parts. In contrast, dihydrobenzoic acid, methylmalonic acid, and kaempferide were key metabolites in the root parts. Aerial parts of A. amoenus showed the highest antiradical and ion reducing properties. Roots of A. tigridis and A. microcephalus, as well as the aerial parts of A. amoenus exerted the highest acetylcholinesterase inhibition activity. Escherichia coli (ATCC 10536) showed the highest sensitivity towards A . yuksellii roots, while its aerial parts displayed significant antifungal activity against Candida albicans (YEPGA 6379), C. parapsilosis (YEPGA 6551), and C. tropicalis (DBVPG 6184). A. microcephalus, A. tigridis, and A. amoenus were toxic to the A549, DU-145, and HELA cell lines but had a low selective index (<1). Conclusion In conclusion, the five Astragalus species studied show strong potential as sources of bioactive molecules for diverse food and pharmaceutical applications.