Rhus ruspolii Engl. plant is traditionally used in Ethiopia to treat various diseases. However, the biological and phytochemical properties of the leaves are not well documented. Hence, this study aimed to isolate phytochemicals from R. ruspolii leaves and evaluate their antibacterial and DPPH radical scavenging activities. GC-MS analysis identified 16 compounds from combined fractions 6–10. Chromatographic separation and NMR analysis resulted in the isolation and characterization of palmitic acid (7), 3,4-dihydroxybenzoic acid (17), cupressuflavone (18), amentoflavone (19), shikimic acid (20), avicularin (21), and myricetin-3-O-5''acetylarabinofuranoside (22). The inhibition zones of extracts (100 mg/mL) and isolated compounds (5 mg/mL) ranged from 8.33 ± 0.50 to 16.33 ± 0.47 mm against all evaluated bacteria. Of all isolated compounds, compounds 18 and 21 showed good activity against Gram-negative (supported by in silico molecular docking studies) and Gram-positive bacteria, respectively. The lowest (49.1 %) and the highest (91.3 %) DPPH radicals were inhibited by combined fractions 6–10 and compound 17, respectively, at 62.5 μg/mL. The SwissADME online analysis showed compounds 17 and 20 have good solubility and permeability. The Pro Tox 3.0 online analysis revealed none of the isolated compounds are fatal if swallowed. Therefore, the findings of this study support the traditional use of the plant for treating bacteria diseases.