Background: Mentha piperita L. essential oils have different antibacterial activity. In the present study, we investigated the effect of &gamma–irradiation on the antibacterial activities of Mentha piperita L. essential oils in vitro and in vivo systems. Materials and Methods: The aerial parts of peppermint were irradiated in a cobalt60 source with 0, 10 and 25 kGy absorbed doses. Then, the plants were subjected to Clevenger extraction to obtain essential oils. The peppermint oils were evaluated for the potential activity against four pathogenic bacteria: Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus in vitro system. The antibacterial assays were determined by using agar well diffusion and disk diffusion methods. In addition, a broth macrodilution method was utilized to evaluate the minimum inhibitory concentrations (MIC’s) of the peppermint essential oils. For evaluation the in vivo antibacterial activity, a surgical procedure by Cecal Ligation and Puncture (CLP) in rats was used. The blood samples were taken from the rats for blood colony forming units (CFU) determination. Results: The results indicated that the oil was effective in reducing CFU caused by sepsis-induced CLP operation. Also, the peppermint oil was effective against all Gram positive and Gram negative organisms tested in vitro system. Irradiated peppermint had no significant antibacterial effects compared with non irradiated one. Conclusion: This study indicated the antibacterial activities of peppermint oils both in vivo and in vitro systems which is sustained even after gamma-irradiation treatment.