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This work focused on screening and characterizing antibiotic-producing Bacillus strains to develop newpeptide antibiotics that can overcome the growing resistance of disease-causing microbes. Twenty bacillus strains were isolated from mud flat using various kinds of Bacillus isolation media. Among them, strain mp56 was determined to show strong antimicrobial activity against multi drug resistant bacteria. 16S rRNA sequence analyses indicated that strain MP56 belonged to the genus Bacillus. The optimal medium composition for production of antimicrobial substance in the Bacillussubtilis MP56 were 1% mannitol, 1% oat meal, 0.01% CaCl2. Strain MP56 exhibited antimicrobial activity against gram positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) and imipenem resistant bacteria. The result suggests that Bacillus strain MP56 produces high quality antimicrobial substance that might be very useful to control varieties of pathogenic microbial growth.