Marine microorganisms have unique physiological properties and novel metabolites such as biosurfactant to live in extreme habitats (e.g. oil polluted environment). The aim of this study was to characterize some biosurfactant-producing bacteria collected from sediment and seawater samples from the Caspian Sea. These bacteria were isolated using enrichment method in ONR7a medium with crude oil as sole carbon source. Five screening tests were used for selection of the bacteria including hemolysis in blood agar, oil spreading, drop collapse, emulsification activity and bacterial adhesion to hydrocarbon test (BATH). The bacteria isolated were identified using molecular methods. Eighteen biosurfactant-producing bacteria (from 44 different colonies isolated) were selected and 9 isolates were screened as predominant biosurfactant producer belonging to Pseudomonas stutzeri (Strain S1-4-2), P. balearica (Strain S1-4-1), Pseudomonas sp. (Strain S2-1), P. stutzeri (Strain R3-2), P. chloritidismutans (Strain K4-1), Achromobacter xylosoxidans (Strain K4-3), Acinetobacter radioresistens (Strain S1-2), Acinetobacter calcoaceticus (Strain K4-2) and Citrobacter freundii (Strain R3-1). The largest clear zone diameters for oil spreading method observed for P. balearica strain S1-4-1 (14 mm). Also, this strain has the best emulsification activity (100 %); surface hydrophobicity (BATH: 77 %) and reduction of surface tension (34.5 mN m-1 ). Thus, this strain selected as the best isolated strains. The results of this study confirmed that there is sufficient diversity of biosurfactant-producing bacteria in the Caspian Sea and the environmental problem can be resolved by application of these bacteria in petrochemical waste water.