Most Ethiopians use medicinal plants/herbs and their products in traditional medicine to treat many human illnesses. Ocimum lamifolium is a typical example in this regard. This study aimed to conduct phytochemical analysis and antibacterial activity tests on the stem extracts of this plant. The powder of the stem of the plant (500g) was subjected to solvent extraction in chloroform/methanol (1:1) and methanol successively. This results in 14.5g (2.9%) and 3.5g (0.72%) crude extracts, respectively. Phytochemical screening tests on the extracts revealed the presence of alkaloids, anthraquinones, flavonoids, glycosides, phenols, saponins, steroids, tannins and terpenoids. The column chromatographic separation of chloroform/methanol (1:1) extract also resulted in the isolation of Stigmasterol (LM-3), Oleic acid (LM-2) and (5Z, 9Z)-22-methyl-5, 9-tetracosadienoic acid (LM-1). Antibacterial activity tests against E. coli, S. aureus, P. aeruginosa and S. pyogenes showed the methanol extract to be relatively more active than chloroform/methanol (1:1) extract. Consistent with literature reports, the isolated compounds (LM-3, LM-2, and LM-1) also showed interesting antibacterial activities against S. pyogenes with superior activity exhibited by compound LM-1. S. aureus and S. pyogenes were susceptible to both isolated compounds and extracts. Though the data were lower than that of the reference drug (Ampicillin), the observed activities could justify the traditional use of the plant to treat different bacterial infections in humans.