Background: In food industry, quality assurance and prolongation of shelf-life is achieved through control of pathogenic and spoilage microorganisms in foods. Regards to much proved adverse effects of synthetic food additives and general concern on the subject, the replacement of these additives with natural based ones such as plant essential oils and natural antibiotics has been raised and researches first at in vitro models and then in food systems is necessary. Objectives: The aim of this study was to determine the antimicrobial activity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Zataria multiflora Boiss. essential oil and nisin alone and in combination against Listeria monocytogen in brain heart infusion (BHI) broth. The specifications of combination of essential oil and nisin were determined. Methods: In this study 600 µg/ml of Z. multiflora essential oil and 2.5 µg/ml nisin in pH value of 5 at 15 ºC were used. Results: In this study Zataria multiflora essential oil had antimicrobial effects agains Listeria monocytogen (MBC: 19, MIC: 9.5µg/ml). Combination of nisin and the essential oil had also antimicrobial effects against Listeria monocytogen (MIC: 1.2, MBC: 2.4µg/ml). Conclusion: Zataria multiflora essential oil and nisin were effective against Listeria monocytogen alone. This effect was obviously increased in combination with each other and the results were noticeable.