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In this study 152 bacterial strains were isolated from the contaminated oils in the southwest of Iran. Hemolysis was used as a criterion for the primary  isolation of biosurfactant producing-bacteria. Fifty five strains had haemolytic activity , among them tweleve strains were good biosurfactant producers by measuring surface tension and emulsification activity.Two microorganism showed the highest biosurfactant production when grown on paraffin and glycerol as the sole carbon source. Glycolipid production by the isolated bacterium using different carbon (n-hexadecane ,  paraffin oil, glycerol, molasses ) and nitrogen sources (NaNO3, (NH4)2SO4 and CH4N2O) was studied. Biosurfactant production was quantified by surface tension reduction, critical micelle dilution (CMD) , emulsification capacity (EC), and thin layer chromatogeraphy. Best result were obtained when using glycerol at a C/N ratio of 55/1 and sodium nitrate as nitrogen source. Production of the rhamnolipid, expressed by rhamnose was 4.2 g/L and the yield in relation to biomass, was Yp/x = 0.65 g/g. Additionally, physical-chemical characteristics of the spent broth with and without cells showed a low critical micelle concentration of 19 mg/L and a decrease in surface tension to 20 mN/m (%).