In vitro cytotoxic, antioxidant, and antimicrobial activities along with total phenolic and flavonoid contents of methanol, hexane, dichloromethane, butanol, and aqueous extracts and chemical composition and fatty acids of n-hexane extract from endemic Hedysarum aucheri were determined. The fatty acid content of the n-hexane extract was determined by GC-MS analysis. Twenty-four out of 39 compounds identified as fatty acid methyl esters constitute 84.20% of hexane extract content. Five of 6 major components were fatty acids, namely alpha-linolenic acid (ALA) (32.37%), palmitic acid (24.69%), linoleic acid (LA) (9.16%), stearic acid (7.11%), Arachidic acid (3.14%), and the other one was an acyclic diterpene alcohol called phytol (12.01%). Butanol extract was found to be the richest in flavonoid (66.3 +/- 1.3 mg QU/g DW) and phenolic (122.4 +/- 1.6 mg GAE/g DW) compounds. Antioxidant activities of the extracts were evaluated by Cupric-ionreducing antioxidant capacity (CUPRAC), and ABTS radical scavenging capacity. Butanol extract showed the highest antioxidant activity indicating a strong correlation between flavonoid/phenolic content and antioxidant activity. Also, cytotoxic activities of the extracts were evaluated against a panel of cancer cells (PC3, HeLa, CaCo-2, U-87MG, A549, and MCF-7) and normal cell line HEK293. Dichloromethane extract (DCM) exhibited exceptionally high tumor selective cytotoxicity towards cancer cells with IC50 values varying between 0.64 and 24 mu g/mL while not impairing the normal cells. DCM extract was found to possess higher cytotoxic activity towards the tested cancer cells than some anticancer drugs reported in the literature. Also, antimicrobial activities of the extracts were determined by agar disc diffusion and the micro-dilution methods against a series of microorganisms and hexane extract showed the highest antimicrobial activity. We hereby report both the pharmaceutical potential of H. aucheri concerning its various biological activities and the phytochemical composition regarding its total flavonoid, phenolic, and fatty acid content.