The main constituents of essential oil (EO) of Origanum syriacum L. collected from cultivated and growing wild in Sinai, Egypt were identified and determined by GC–MS. The antimicrobial activity of these essential oils against Gram positive and Gram negative bacterial strains was studied. The antimycotic activity of these EOs was also evaluated against eight fungal strains isolated from different sources. Forty-six compounds were identified in the EO of both samples, dominated by carvacrol in cultivated type. Thymol, γ-terpinene, linalool and 4-terpineol were the most represented constituents in O. syriacum plants collected from wild populations. Both EOs showed antibacterial activity with varying magnitudes, while EO from cultivated O. syriacum showed high antibacterial activity against Staphylococcus aureus with an inhibition zone of 32 ± 4.0 mm. Both EOs showed good antifungal activity against all fungal strains. O. syriacum EO from cultivated plants showed the lowest MIC 0.25 mg/L with Aspergillus fumigates clinical strain isolated either with Aspergillus flavus.