Members of the Bacillus genus are considered as the factories for the production of biologically active molecules that are potential inhibitors of growth of pathogens.Antimicrobial lipopetides have the potential to play an important role in preventing the rapid development in bacteria resistance to conventional antibiotics.Bacterial strains were isolated from poultry intestines and then screened to identify the Bacillus genus producing surfactins. These molecules were purified and characterized using ESI MS / MS.Finally, the antibacterial effectiveness of these surfactins against C. perfringens was assessed.A poultry intestinal Bacillus mojavensis5RCN with broad-spectrum antibacterial activity has been identified. The successful extraction of an effective antimicrobial compound from the fermentation media has been accomplished.During the primary screening, a strain that produced antimicrobial lipopetides and exhibited significant hemolytic activity, was isolated and identified. This identification was achieved through genotypic and morphological characterization. Moreover, ESI MS/MS analysis of the antibacterial molecule showed that it contained cyclic lipopeptides with C13, C14, C15, C16, and C17 acyl chains, and revealed the occurrence of most abundant surfactin homologues (1006, 1021, 1034, 1048, and 1062 m/z). The isolated surfactin displayed potent antibacterial activity against Clostridium perfringens. In light of this study, Bacillus mojavensis5RCN strain and its effective antibacterial surfactin lipopeptide could be utilized in a variety of food production and could have promising medical applications.