In present investigation, B. subtilis and B. cereus was isolated and Identified from the petroleum contaminated site soil and identified by gram’s staining, motility and biochemical tests. After the isolation and identification, biosurfactant production from bacterial organisms was screened using mineral salt medium, by oil spreading, drop collapse and hemolysis test. From the blood agar plates, the biosurfactant producing bacterial culture were obtained and maintained as stock cultures. Organisms were inoculated into the mineral salt broth with petroleum oil, diesel oil, kerosene oil and crude oil as a carbon source for biosurfactant production. Biosurfactant was extracted by acid precipitation method. Biosurfactants production was optimized by using different pH, temperature, carbon and nitrogen sources. Emulsification activity of biosurfactant was analyzed. The extracted biosurfactant was characterized by using Thin Layer Chromatography (TLC). B.subtilis was able to produce the biosurfactant using pH (4), temperature (20oC), Carbon source (glucose), nitrogen source (NaNO3). B.cereus was able to produce the biosurfactant by using pH (6), temperature (35oC), carbon source (Starch), nitrogen source (urea). Among this study, Bacillus subtilis produced higher amount of biosurfactant than Bacillus cereus.