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Astragalus species are ubiquitous in the pharmacopeia of numerous countries, signifying their widespread medicinal applications. Türkiye is esteemed as one of the principal epicenters of diversity for this genus with 483 taxa, and many of these plants have been traditionally utilized for diseases including coughing, diabetes, cardiovascular disorders, and aches. Although there is a considerable body of chemical and biological research available on several Astragalus species, studies focusing on Astragalus brachystachys DC are exceedingly rare. In this context, This study provides the first comprehensive report on this medicinal plant the chemical constituents and biological activities of an important medicinal plant, Astragalus brachystachys DC. The aerial part samples were collected from Adana, Türkiye, and an ethanol extract was prepared with these parts. The secondary metabolites of the extract were determined by an LC-HRMS analysis. The LC-HRMS analysis showed the presence of 39 different constituents, hyperoside (303.419±10.50 μg/g extract), p-coumaric acid (256.975±8.51 μg/g extract), and rutin (72.684±2.23 μg/g extract) were determined as major compounds in the aerial parts ethanol extract. Attributed to its high total phenolic (58.53±1.30 μg PEs/mg extract) and total flavonoid content (29.98±0.83 μg QEs/mg extract), the extract demonstrated strong antioxidant activity according to three different assays namely DPPH free (IC 50 : 33.08±0.61 μg/mL), and ABTS cation radical scavenging (IC 50 : 15.39±0.72 μg/mL) and CUPRAC activity (A 0.5 : 36.25±0.28 μg/mL) methods. In vitro assays showed that cholinesterase inhibitory activity results were found to be exceptional with 85.95±0.52 % inhibition on acetylcholinesterase and 66.32±1.33 % inhibition on butyrylcholinesterase at 200 μg/mL. Regarding antimicrobial properties, Astragalus brachystachys DC extract was found to be effective against Enterococcus faecalis with a MIC value of 39.06 μg/mL., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)