Objectives: Zataria multiflora Boiss has been used as a conventional medicinal plant for treating infections in traditional medicine. Therefore, the aim of this work was to reveal the composition and antibacterial effect of Zataria multiflora leaves essential oil. Materials and Methods: Z. multiflora essential oil was isolated through two different methods: hydro isolation and steam isolation. Essential oil was analyzed by gas chromatography–mass spectrometry (GC/MS) and its composition was determined. The antibacterial effect of Z. multiflora essential oil was investigated on gram-positive (Staphylococcus aureus, Streptococcus mutants, Staphylococcus epidermidis) and gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium) bacteria compared to that of 12 antibiotics. Results: The essential oil yields in hydro isolation and steam isolation methods were 2.33% and 0.33% respectively. Thirtyseven compounds were identified in the essential oil using GC/MS, among which carvacrol, thymol, linalool, p-cymene, decane, β-caryophyllene and γ-terpinene were the dominant compounds. Conclusions: Z. multiflora essential oil, at the concentration of 80 µL/mL, had a strong or similar antibacterial effect on S. aureus and S. typhimurium compared to the antibacterial effect of some of the antibiotics in the study.