Rubus apetalus Poir. is a medicinal plant used in Ethiopia to treat different diseases. This study aimed to isolate phytochemicals from the leaves and evaluate their antibacterial and DPPH scavenging activities using in vitro and in silico studies. The GC-MS study resulted in the identification of 16 compounds. Palmitic acid (9), palmityl alcohol (17), β-sitosterol (18), tiliroside (19), and astragalin (20) were isolated using chromatographic techniques and characterized using NMR data. The inhibition zones (IZs) of 100 mg/mL extracts and 5 mg/mL isolated compounds ranged from 9.33 ± 0.47 to 15.07 ± 0.74 mm against all bacteria. Compound 18 showed better activity against Staphylococcus aureus and Streptococcus pyogenes. Compound 19 exhibited better activity against Escherichia coli and Pseudomonas aeruginosa. The activity of compound 19 was supported by molecular docking studies against E. coli DNA gyrase B (−8.0 kcal/mol) and Pseudomonas quinolone signal A (PqsA, −9.5 kcal/mol) for E. coli and P. aeruginosa, respectively. At 62.5 mg/mL, combined fractions 16–21 and compound 19 showed the lowest (54.5 %) and the highest (89.8 %) DPPH scavenging activity, respectively. The in silico studies indicated that none of the isolated compounds is fatal if swallowed. Therefore, these findings support the traditional use of the plant to treat various bacterial diseases.

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