Background: Microorganisms resistant to most antibiotics are rapidly spreading, and there is an urgent and continuous need for novel antimicrobial compounds. The genus Citrus belongs to the family Rutaceae has many biologically active secondary metabolites. Objectives: The purpose of this study was to evaluate antimicrobial activity of essential oil and extract of Lemon (Citrus limon), Mandarin (Citrus reticulata) and Pummelo (Citrus grandis) against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella typhi. Materials and Methods: The fresh Citrus leaves were shade-dried and powdered. Antimicrobial metabolites were extracted from them by 80% methanol for extract and using a Clevenger-type apparatus for essential oil. Eight different concentrations of the each leaf extract and essential oil were prepared. The antimicrobial susceptibility assay of Citrus leaves metabolites were subjected against four bacterial strains by agar disc diffusion and E-test method. Results: In this study, minimum inhibitory concentrations (MIC) of different Citrus leaf extracts were determined against all four food-borne pathogens. The C. grandis leaf essential oil had potent antimicrobial activity against all four pathogens, and the C. limon leaf essential oil was effective on Gram-positive bacteria. S. typhi was resistant against two leaves essential oils. Conclusions: The results showed that there was no antimicrobial activity effect in all extracts on tested bacteria. In this study, the antibacterial effect of essential oil of Citrus leaves on four strains of pathogenic microorganisms was confirmed. The C. grandis leaf essential oil had the most powerful antimicrobial properties, suggesting its potential application as natural preservative in foods or an effective medicine against different pathogenic microbes. Key words: Antibacterial activity, E-test, Citr