This study aimed to enhance the removal of petroleum with potent biosurfactant producing bacterial strains (Bacillius subtilis CC9, Enterobacter asburiae WMB1, Klebsiella oxytoca Rizhao 536-1, and Acinetobacter pittii PgBE252) isolated from an oil field. The biosurfactant production was determined by different screening methods such as hemolytic activity, drop-collapse, oil spreading, BATH, emulsification index and surface tension. While the presence of biosurfactant was detected with individual bacterial strain, B. subtilis CC9 strain was found to be quite effective on petroleum degradation (92%) with a high yield of biosurfactant production (0.95 ±0.05 g L-1). The degradation rate constant and half-life period of B. subtilis CC9 strain were calculated as K = 2.533 day−1, t1/2 = 0.273. It has been clearly shown with GC/MS analysis that B. subtilis CC9 strain was effective on the removal of long chain n-alkanes (C31 - C35) as well as short and medium chains. The FT-IR analysis also approved the presence of aliphatic and peptide moieties of biosurfactant extracted from B. subtilis CC9 strain. It is remarkably pointed out with this study that high yield of biosurfactant producing B. subtilis CC9 strain can be used in advanced petroleum bioremediation studies as an alternative to chemical surfactants.