Petroleum hydrocarbons are the most common environmental pollutants and oil spills pose a great hazard to terrestrial and marine ecosystems. In the current study two oil-contaminated water samples from Gemsa Company and Adabiya Suez Port were collected. Some different physico- chemical properties of the contaminated water samples were characterized. The extraction and characterization of oil from studied samples were performed. Nine bacterial strains were isolated from the contaminated samples. The bacterial isolates were screened for the production of biosurfactants. Two promising bacterial strains (B3 and B4) were selected from bacterial isolates according to the highest oil displacement test, emulsification power and the lowest surface tension results. The biosurfactants were recovered from the cultural media of (B3 and B4) at 1.1 and 1.3 g/L respectively. These bacterial isolates were identified using molecular identification performed by 16S rRNA gene sequences. The results indicated that the two isolated bacteria (B3 and B4) belong to Bacillus flexus and Gelidibacter japonicus respectively. The characterization of the produced biosurfactants were determined by FTIR analysis. The result of FTIR analysis, indicated that the biosurfactants obtained from Bacillus flexus and Gelidibacter japonicus are mainly a complex lipopeptides with a small fraction of glycolipids called polymeric biosurfactants.