The present study deals with laterosporulin biosynthesis from newly isolated Brevibacillus sp. in liquid culture. Soil samples were collected from different habitats of Lahore(Pakistan) for the isolation of bacterial cultures. After primary and secondary screening, ISL-4 (0.53 IBU/ml) and ISL-7 (0.6 IBU/ml) were selected having better potential for bacteriocin. A significant (p≤0.05) increase in laterosporulin biosynthesis was observed when the parameters like medium volume, buffer pH (7.0) and incubation temperature (37°C) were optimized. Antimicrobial potential of laterosporulin was also exploited using various techniques. Minimum inhibitory concentration (MIC) and critical dilution assays (CDAs) were performed for exhibiting bacteriocin inhibitory potential against various indicator strains including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Azotobacter sp. It was concluded that Brevibacillus sp. possesses strong potential for laterosporulin biosynthesis. Further manipulation of physical and nutritional parameters improved its biosynthesis in liquid culture.