As a herbal medicine, essential oil is used for the treatment of several chronic diseases, however, the clinical evidence of essential oil is insufficient. In this context, we have made an effort to quantify its percentage of chemical composition and the biological potency of essential oil of the leaves of Psidium guajava. The content of essential oil was found in 0.38% (v/w) and the oil was isolated by using hydro distillation from the leaves of the selected plant. The isolated oil was analyzed by using gas chromatographyâmass spectrometry (GCâMS) equipped with a HP-5MS fused silica capillary column (30 m Ã 0.25 i.d., film thickness 0.25 μm). The antimicrobial and cytotoxic activities of the isolated leaves oil at different concentrations were determined by agar gel diffusion and brine shrimp lethality bioassay. The antimicrobial activity of the essential oil at various concentrations was determined against three Gram (+) Enterococcus faecalis, Staphylococcus aureus and Streptococcus aureus and three Gram (â) Escherichia coli, Haemophilus influenzae, and Pseudomonas aeruginosa bacterial strains. A total of 54 chemical compounds were detected by using GCâMS which is representing 98.172% of the total oil. The dominating ingredients in the leaves oil were iso-caryophyllene (33.53%), veridiflorene (13.00%), farnesene (11.65%), dl-limonene (9.84%), δ-cadinene (1.75%), α-copaene (2.80%), α-humulene (3.74%) and Ï-cadinol (0.08%). The oil showed significant antimicrobial activity against both Gram (+ and â) bacterial strains with an inhibition range of 0â13 mm. However, the isolated leaves oil did not show any activity at any concentration against brine shrimp lethality bioassay. The isolated oil showed significant antimicrobial activity and it could be used as natural antibiotics.