Background and Objectives: Biosurfactants are a group of amphipathic molecules produced by microorganisms, which are constructed from two portions: hydrophilic and hydrophobic; these compounds increase biodegradation of insoluble pollutants. This study was carried out to isolation, identification and characterization of biosurfactant-producing Shewanella species from the Persian Gulf.Material and Methods: In this study, 25 biosurfactant producing strains were isolated from polluted sediments and seawater from Persian Gulf. Biosurfactant producing bacteria were isolated in Bushnell-Hass medium enriched with diesel. Then, dominant strains were screened by using quantitative and qualitative tests, including hemolysis in blood agar, oil spreading, drop collapse, emulsification activity and BATH test. Probable strains were identified by biochemical methods. Finally, dominant biosurfactant producing bacteria were recognized by using universal primers for 16s rRNA and sequencing.Results: In total of seven biosurfactant producing bacteria were isolated. Two of them, Shewanella E14 and Shewanella N4, were identified as predominant strains. Also, Shewanella algae and Shewanella upenei confirmed by molecular method.Conclusion: Based on the results, the isolates were able to produce biosurfactants and at the same time were able to degrade hydrocarbons. Therefore, the evaluation of applied potential of these strains for bioremediation of oil spillages is recommended.