Background: In Turkey, Astragalus species are highly appreciated for their traditional therapeutic values. This research was devised to investigate the chemical composition, antioxidant, and enzyme inhibition activities of the aerial components of Astra-galus (A.) angustifolius subsp. pungens, A. prusianus, and A. zederbaueri. Methods: Four extracts namely; ethyl acetate (EtOAc), methanol (MeOH), 70% MeOH, and aqueous extracts, were meticulously prepared. The chemical composition was examined utilizing spectrophotometric and High performance liquid chromatography-mass spectrometry (HPLC-MS)/MS techniques. The antioxidant activity was assessed by investigating the extracts’ capability to scavenge radicals, reduce ions, and chelate ferrous ions. The enzyme inhibition properties were evaluated against acetyl-cholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase (Tyr), α-glucosidase, and α-amylase enzymes. Results: The findings revealed that the greatest total phenolic (28.33 mg gallic acid equivalent/g) and flavonoid (66.59 mg rutin equivalent/g) contents were achieved with the MeOH extract of A. angustifolius. The chemical profiles of the three species were marked by the presence of flavonol glycosides containing kaempferol, rhamnetin, isorhamnetin, and quercetin structural motifs. MeOH extract of A. angustifolius exerted the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (48.73 mg trolox equivalent (TE)/g), Cu++ (150.10 mg TE/g) and Fe+++ (84.78 mg TE/g) reducing capacity. The three polar extracts (67.18–69.64 mg TE/g) of this species in addition to the aqueous extract (69.46 mg TE/g) of A. zederbaueri displayed the best 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. 70% MeOH extract of A. prusianus had the highest metal chelating power (25.61 mg ethylenediaminetetraacetate equivalent (EDTAE)/g). MeOH extracts (2.37–2.50 mg galatamine equivalent (GALAE)/g) of the three species revealed the highest AChE inhibition activity while EtOAc extract (2.01 mg GALAE/g) of A. zederbaueri had the highest BChE inhibition activity. The highest anti-Tyr (50.40 mg kojic acid equivalent/g) activity was recorded from the EtOAc extract of A. zederbaueri. Conclusion: Thus, the three species of the studied Astragalus may represent a promising natural source of pyhtopharmaceutical agents for various pharmaceutical and nutritional uses.