Background and Objective: Indiscriminate use of antibiotics has resulted in resistant microorganisms. In order to find new antibiotics to defense pathogenic microorganisms resistant to common antibiotics, An antibiotic-producing bacterium isolated from skin of the frog Rana ridibunda.Material and Methods: In sterile conditions samples from skin of frog were transferred to broth medium. Antimicrobial agent-producing bacteria were isolated using serial dilution method on nutrient agar. One of them was selected for more investigation. Isolated bacterium identified based on 16SrDNA and biochemical and morphological characteristic. Antimicrobial activity of culture supernatant was examined against laboratorial standard Bactria by disc diffusion and MIC methods. In order to characterization of produced Antimicrobial compound, Culture supernatant of bacterium was washed by chloroform and then Antimicrobial substance was extracted by methanol and chloroform and detected by bioautography on silica gel plates. The dialysis tube was used to find the molecular weight of this substance.Results: Isolated bacterium identified as strain of Bacillus atrophaeus. The anti-microbial substance exhibited heat stability between 25 and 100°C, and activity under both acidic and basic conditions from pH 2 to 11. Bioauotography assay showed that methanol is optimum solvent for the extraction. The dialysis tube indicated that the Antimicrobial substance weight is less than 1 kDa and Ammonium sulfate was not capable to precipitate the compound.Conclusion: This study showed that Antimicrobial substance differs from peptide antibiotics produced by bacteria of the genus Bacillus such as bacitracin, in some properties that increases the likelihood of being novel.