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Aims: Antimicrobial effect of Allium akaka herbal extracton the formation of biofilm and colonization of clinical and food-born pathogens are important in this research. Study Design: In the recent years, traditional medicine and identification of herbs have been highly addressed due to the problems arising from consumption of chemical drugs and bacterial resistance to the antibiotics in planktonic and biofilm modes. Allium (with the scientific name Allium akaka) contains sulfide and sulfur compounds with strong antimicrobial effects. Place and Duration of Study: Islamic Azad University of North Tehran Branch Laboratory, between February 2012 and May 2013. Methodology: Efforts have been made in this research to study the inhibitory effect of Allium ethanol extract on Staphylococcus aureus and Salmonella typhimurium bacteria using disk diffusion and macro dilution methods (concentrations 62.50, 125, 250 and 500 mg lit -1 ). Also in this experimental research, the effects of different concentrations of Allium extract on biofilm formation bacterial and S. aureus and S. typhimurium pathogenic bacteria were studied using micro-titer method. Results: The best effect of extract was for S. typhimurium bacterium which was estimated Original Research Article Annual Research &Review in Biology, 4(20): 3087-3095, 2014 3088 at 125mg lit -1 . The lowest dilution for Allium herbal extracts that impeded the formation of biofilm in both S. aureus and S. typhimurium bacteria included a dilution of the 0.55 gr ml -1 within the first 24 hours and had lower effects on non-formation of biofilm within the next 48 and 72 hours. Conclusion: The present study shows that ethanol extract of A. akaka has antibacterial activity against S. aureus and S. typhimurium bacteria also the ability to diminish the