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Two Bacillus sp. namely B. amyloliquefaciens SAS-1 and B. subtilis BR-15 isolated from two different sites were employed in biosurfactant production. The current work describes the statistical optimization of biosurfactant production and comparative characteristics study of lipopeptides produced by these microbial cultures. The composition of production medium and culture conditions were optimized to enhance the lipopeptide yield using response surface methodology (RSM). For each strain, the experiments were conducted in two sets as per designed by Central Composite Rotatable Designs (CCRD). The surface quadratic models obtained with these experimental results were validated by analysis of variance (ANOVA) and were found to be significant with F-test. Under optimized conditions, lipopeptides yield was increased from 1.13 and 1.72 g/L to 2.08 and 2.40 g/L respectively with strains SAS-1 andBR-15 …