Background: Lavandula genus, the member of Lamiaceae family comprises thirty known species among which Lavandula angustifolia is economically important in toiletries and perfumery industries. L. angustifolia is commonly known in Iran as “Ostokhoddous” and is rich of essential oil. L. angustifolia oil has several medicinal and biological properties such as antispasmodic, carminative, diuretic, sedative, anti-inflammatory and analgesic, anthelmintic agent.Methods: The aim of this research was to evaluate the chemical composition of L. angustifolia oil by GC and GC-MS and its antimicrobial activity by disc diffussion and microbroth dilutution in vitro condition.Results: GC and GC-MS analysis of essential oil showed the presence of 1, 8-cineole (34.8%), borneol (24.6%), camphor (10.4%) as the main components. The antimicrobial evaluation by disc diffusion and microbroth dilution against S. aureus, S. saprophyticus, B. cereus, E. coli, S. typhimurium, C. albicans, A. niger exhibited that B. cereus (IZ=18.4 mm, MIC and MLC= 2, 4 ml/ml), S. aureus (IZ=14.9 mm, MIC and MLC= 2, 2 ml/ml), S. saprophyticus (IZ=13.0 mm, MIC and MLC= 2, 4 ml/ml) were susceptible microorganisms, while C. albicans (IZ=10.5 mm, MIC and MLC= 8, 16 ml/ml), gram negative bacteria (IZ=7.0-8.0 mm, MIC and MLC= 2-8; 4-8 ml/ml) and A. niger (IZ=7.0 mm, MIC and MLC= 16, 32 ml/ml) were less sensitive microorganisms than gram positive bacteria.Conclusion: From this explanation, the present antimicrobial activity of L. angustifolia oil against gram positive bacteria is related to its main components and because of lower antimicrobial of this component compared to phenolic compounds such as thymol or carvacrol, L. angustifolia oil appears as a medium antimicrobial agent.