The objective of this study was to determine the chemical composition as well as antioxidant, antibacterial, and cytotoxic properties of the essential oil of Mentha piperita L. (peppermint). Fifteen chemical constituents were identified in the essential oil, for a total of 99.99% of the compounds. The essential oil exhibited antimicrobial activity against two Gram-positive bacteria Staphylococcus aureus and Listeria monocytogenes. The minimum inhibitory concentration (MIC) of essential oil of Mentha piperita L. for Staphylococcus aureus and Listeria monocytogenes was 1.84 μg/ml, whereas the minimum bactericidal concentration (MBC) values were 3.7 and 7.43 μg/ml, respectively. The oil displayed potent antioxidant activity inhibiting up to approximately73% of 2,2ʹ-azinothiobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals. In the cytotoxicity assay, the highest essential oil concentration (100 μg/ml) resulted in viability of approximately 90% human epidermal keratinocyte (HaCaT) cells. With respect to antitumor activity in C6 rat glioma cells, there was significant reduction in cell viability: 56–74% in 24 hr, and 71–77% in 48 hr. Data suggest that in presence of the essential oil of Mentha piperita L. antioxidant, antibacterial, antitumor and non-cytotoxic properties were noted.