Hyssopus officinalis L. has been traditionally used for its antiseptic properties in treatment of infectious disorders. In order to confirm its antiseptic properties with respect to traditional uses, we have screened the antimicrobial activity of aerial parts of H. officinalis L. essential oil against different microorganisms. The oil was obtained using hydro distillation and analyzed by GC and GC-MS. Antimicrobial activity of H. officinalis L. essential oil was screened by disc diffusion and micro broth dilution assays.Analysis of essential oil revealed the presence of isopinocamphopinone (39.3 %) and isopinocamphone (22.1%) as the major components. The results exhibited significant activity against Gram-positive bacteria with inhibition zone and minimal inhibitory concentration value in the range of 7-16 mm and 0.5-1 μl/ml,respectively. Moreover, the oil showed inhibitory effect against fungi. This work confirmed the traditional uses of H. officinalis as antimicrobial agent for treatment of infectious disorders. Further clinical trials are required to validate its uses as therapeutic, alternative agent for treatment of infections.