The leaves of Eucalyptus globulus were steam distilled to isolate the essential oil and the characterization was done by GC–MS. The identification of thirty-two compounds was confirmed that represented 100% of the oil. The key components identified were 1,8-Cineole (80.20%) α-Pinene (8.5%), β-Eudesmol (3.44%) α-Phellandrene (1.09%). The antibacterial activity of the oil was tested by the disc diffusion and micro-broth dilution methods against two bacterial isolates. The oil exhibited moderate growth suppression against both the Gramnegative bacteria with inhibition zones of with inhibition zones of 16 to 19 mm and MIC values of 125 to 500 μg/mL. 1,8-Cineole and β-Eudesmol were found to be the active antibacterial compounds present in the oil.