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. The lipopeptides produced by both strains were equally efficient to emulsify (~68.0%) crude oil, nevertheless better than chemical surfactants. Though, the lipopeptides of strain BR-15 were more effective in terms of yield and crude oil displacement (2.40 g/L and 36.298 ± 2.548 cm2) as compared to those of strain SAS-1 (2.08 g/L and 30.183 ± 3.781 cm2). The characteristics difference can be attributed to the production of different isoforms of surfactin (lipopeptides) by B. amyloliquefaciens SAS-1 and B. subtilis BR-15. Further, the emulsification and oil displacement efficiency of lipopeptides highly recommend these as potentially applicable in bioremediation, oil recovery enhancement or as emulsifiers.