AbstractWithania adpressa(Coss.) Batt. (W. adpressa) is a wild medicinal plant in the family Solanaceae, which is used as an alternative medicine. The present study aims to investigate the chemical composition, antioxidant, and antibacterial potentials of polyphenol-rich fraction from the leaves ofW. adpressa. Polyphenol-rich fraction was characterized by use of high-performance liquid chromatography (HPLC). Antioxidant potency was determined by use of 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and total antioxidant capacity (TAC) assays. Antibacterial activity was assessed against gram-positive and gram-negative bacteria by use of disc diffusion and microdilution assays. Chromatographic analysis by HPLC showed four compounds in the polyphenol-rich fraction including 1-O-Caffeoyl glucose, Luteolin-7-diglucuronide, Apigenin-O-pentoside, and Quercetin-3-O-glucuronide. Polyphenol-rich fraction exhibited important antioxidant activity as assessed by DPPH and FRAP assays, with IC50and EC50values of 14.27 ± 0.43 and 39.48 ± 0.81 µg/mL, respectively, while butylated hydroxytoluene (BHT) and Quercetin exhibited IC50values of 28.92 ± 0.24 and 22.48 ± 0.54 µg/mL. Results of TAC showed that the polyphenol-rich fraction scored 781.74 ± 2.83 µg AAE/mg. Polyphenol-rich fraction showed an important antibacterial effect vs gram-positive and gram-negative strains recording inhibition zone diameters varying from 19.5 to 29.2 mm, while antibiotics were almost ineffective toward all strains except forE. coli. It can be concluded thatW. adpressapolyphenol-rich fraction possesses promising phenols with strong antioxidant and antibacterial properties, which may help fight pathogenic bacteria and free radicals.