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In the search for bioactive metabolites from Ethiopian medicinal plants, two tetracyclic triterpenes; spinasterol (1) and shionone (2) along with cetyl alcohol (3) were isolated from the acetone extract of the stem bark of Vernonia biafrae, a medicinal plants widely used for the treatment of infectious diseases. Column chromatographic separations and Sephadex LH-20 gel filtration were employed for the isolation of the pure compounds. The compounds were characterized by spectrometric (NMR and mass) analyses and comparison with literature data. This is the first report of compound 2 and 3 from the genus Vernonia. The extracts and the isolated compounds were evaluated for their antimicrobial activities against both bacterial and fungal test strains and exhibited comparable activities with the standard antibiotics. The highest activity was observed for acetone extract (23.73 0.10 mm) and compound 2 (23.72 0.30 mm) against Staphylococcus aureus. Whereas, the rest showed moderate to low inhibitory activities against the test strains. The finding could be used for comprehensive evaluations of the phytochemicals for their microbial activities and also support the claim that the plant V. biafrae is used for the treatment of microbial diseases.