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Introduction & Objective: Rosmarinus officinalis L. as a member of the Lamiaceae family and lysozyme as a natural antibacterial agent is important in food microbiology, because of its characteristics. The aim of the present study was to determine the chemical composition and anti-listerial activity of Rosmarinus officinalis essential oil (REO) alone and in combination with lysozyme for enhancement of anti-listerial activity of both substances. Materials & Methods: Rosmarinus officinalis L. was purchased from a local grocery store at Shahrekord and was identified by the Institute of Medicinal Plants, ACECR. The air-dried aerial parts were subjected to hydrodistillation using a Clevenger apparatus to obtain essential oil and yielded oil was analyzed by GC/MS. Antibacterial activity (on basis of Minimum Inhibitory Concentration (MIC) of REO was studied separately and in combination with unheated lysozyme (L) and heat-treated lysozyme (HTL) on Listeria monocytogenes at different pH (5, 6 and 7) by a micro-broth dilution assay. The collected data were analyzed by SPSS software. Results: In the current study, 98.05% of constituents of the essential oil were identified. The major components were α-pinene (14.06%), 1,8-cineole (13.62%), verbenone (11.2%), camphor (10.51%), borneol (7.3%), 3-octanone (7.02%), camphene (5.46%) and linalool (5.07%). The inhibitory action of REO was stronger at lower pH especially 5 (MIC=225 μg/mL). Inhibition by L at pH 5 was 640 μg/mL but no inhibition was seen at pH 7. HTL resulted in more effective inhibition than L, especially at pH 5 and heat-treatment 80˚C (MIC: 160 μg/mL). Conclusion: Combination of L + REO and particularly HTL + REO was led to enhancement of bacterial inhibition. It was concluded that REO by the identified chemical composition was effective alone or in combination with L or HTL on Listeria monocytogenes as a food-borne pathogen.