Objective: The current study is designed to evaluate the activity of essential oil of Origanum vulgare against multi-drug resistant bacteria and both G+ and G- bacteria. Material and Methods: O. vulgare essential oil was isolated by Clevenger-type apparatus according to the procedure described in the British pharmacopeia volume II. Five species of bacteria were used to evaluate the antibacterial activity of O. vulgare constituent in this study, Two Gram negative (Escherichia coli, and multidrug resistance Klebsiella pneumoniae) and three Gram positive bacteria (Streptococcus pneumonia, Staphylococcus aureus and Enterococcus faecalis). All bacteria isolated from different clinical sources. The stock solution (1-mg /16 mL) concentration had been prepared as follow: 1 mg of essential oil dissolved in 16 mL of (DMSO) dimethyl-sulfoxide. The serial dilution of 1-mg/mL was the following concentrations: 1000, 500, 250, 125 and 62.5 μg/mL were prepared for antibacterial activity by agar well diffusion. Ampicillin and Amoxicillin were used as positive control for all strains, while DMSO as negative control. Conclusion: The essential oil of O. vulgare shows a good antibacterial activity against MDR bacteria as well as the other studied types of bacteria.