Hyssopus officinalis L.has been known as a culinary and medicinal herb for hundreds of years. The present study was designed to investigate the chemical composition, antimicrobial and free radical-scavenging activity of H.officinalis ethanolic extracts which is cultivated in Iran and has not been studied previously. Forty four compounds were identified by gas chromatography-mass spectrometry analysis. The ethanolic extract of aerial parts of Hyssopus officinalis which is cultivated in Iran inhibited the growth of some of examined microorganisms including different kinds of bacteria. The highest antimicrobial activity was observed against Bacillus cereus and Pseudomonas aeruginosa but the extract was inactive against the fungi, C.albicans and A.niger. The ethanolic extract of aerial parts of Hyssopus officinalis was evaluated as DPPH free-radical scavenger. The antioxidant potential of extract was evaluated by FRAP assay and its ability to inhibit lipid peroxidation was also determined by TBARS test in rat. In vivo evaluation of antioxidant effects of H.officinalis with these two methods showed that the extract of H.officinalis has a moderate ability to inhibit lipid peroxidation and has a moderate antioxidant power with all doses comparing to control (p<0.05).