Background and Objective: Probiotics are beneficial organisms therapeutic within microbial flora. Shigella, Escherichia coli and Salmonella are the most common cause of intestinal infectious diseases that lead to morbidity and mortality in infant and children worldwide. The aim of this study was to evaluate anti-microbial activity of Lactobacillus acidophillus and Lactobacillus ruteri against entero-pathoges by in vitro and in vivo methods. Methods: In this experimental study, the therapeutic effect of the lactobacillus acidophilus ATCC 4356 and ruteri ATCC 23272 against Shigella sonnei ATCC 9290, Escherichia coli ATCC 25922 and Salmonella enterica BAA-708 were evaluated by in vitro (spot agar) and in vivo (BALB/c mice) methods. Weight improvment and survival rate in mice were recorded. Results: Lactobacillus acidophillus and ruteri had protective and therapeutic effect against diarrhea caused by pathogenic bacteria. Probiotics reduced the weight, colonization of pathogens and increased the survival rate of animals (P<0.05). Conclusion: Lactobacillus acidophillus and ruteri has anti-microbial activity and their consumption can be effective in the prevention and also the treatment of intestinal disease.