This study was done to extract hydrocarbon compounds from El-Lajjun oil shale using biosurfactant produced from two strains Rhodococcus erythropolis and Rhodococcus ruber. The results have shown that, optimal biosurfactant production was found using naphthalene and diesel as a carbon source for R. erthropolis and R. ruber, respectively. Optimum nitrogen concentration was 9 g/l and 7 g/l for R. erthropolis and R. ruber, respectively. Optimum K2HPO4 to KH2PO4 ratio, temperature, pH, and agitation speeds were 2:1, 37 degrees C, 7 and 200 rpm. Under optimal conditions R. erthropolis and R. ruber produced 5.67 and 6.9 g/l biosurfactant, respectively. Maximum recovery of oil achieved with hydrogen peroxide pre-treatment was 25% and 26% at biosurfactant concentration of 8 g/l and 4 g/l for R. erthropolis and R. ruber, respectively. The extent desorption of hydrocarbons from the pre-treated oil shale by biosurfactant were inversely related to the concentration of high molecular weight hydrocarbons, asphaltenes compounds. Pre- treatment of oil shale with hydrogen peroxide produced better improvement in aromatic compounds extraction in comparison with improvement which resulted from demineralization of the oil shale. (c) 2009 Published by Elsevier Ltd.