The current study was performed to determine the phenolic compounds composition, antioxidant and antimicrobial activity, and α-glucosidase inhibitory effect of the root extract of Tupistra nutans Wall., in order to validate its use in traditional medicine. HPLC-MS/MS analysis showed the presence of ferulic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, salicylic acid, chlorogenic acid, caffeic acid, l-phenylalanine as the predominant phenolic compounds. The antioxidant potentials of T. nutans were determined in different solvent extract and found to vary in a concentration-dependent manner. Ethyl acetate-soluble fraction exhibited the highest amount of phenolic acids, flavonoids, antioxidant activity against the DPPH, reduced Fe 3+ /ferric cyanide complexes to the ferrous form and inhibitory effect on α-glucosidase. Butanol fraction showed the strongest ABTS radical scavenging ability. Methanolic root extract showed higher reduction capability of ferric ions than the other solvent fractions. A significant and higher positive correlation was found between total phenolic content and total flavonoid content with α-glucosidase inhibition and DPPH using ethyl acetate solvent than the other solvent fractions. All the tested microorganisms: Staphylococcus aureus Rosenback (ATCC13150), Salmonella enterica typhimurium Kauffmann and Edwads (ATCC14028), Escherichia coli Castllani and Chalmers (ATCC35150), and fungi: Candida albicans Robin and Berkhout (KTCC7965), Aspergillus fumigatus Fresenius (KTCC6145), A. flavus var. flavus Link ex Fries (KTCC6143), A. niger van Tieghem (KTCC6317) were susceptible to the root extracts at a concentration of 0.12-0.25 mg mL −1. This study may allow us to understand indigenous medicinal values of T. nutans. Furthermore, these results showed that T. nutans is a good source of antioxidants along with antimicrobial and antidiabetic activities and could be used as an important bioresource for the pharmaceutical and food industries.