In this study, 26 Pseudomonas spp. were isolated from a stream polluted by factory waste and from petroleum-contaminated soil. The surface tension (ST) of the cultures was used as a criterion for the primary isolation of biosurfactant-producing bacteria. Biosurfactant production was quantified by ST reduction, critical micelle concentration (CMC), emulsification capacity (EC), and cell surface hydrophobicity (CSH). Two of the isolates, P. aeruginosa 78 and 99, produced rhamnolipid biosurfactant. The strains started rhamnolipid production in the logarithmic phase. They decreased the ST of the culture from 73 dyne/cm2 to 29 and 33 dyne/cm2, and the CMC of produced rhamnolipids were 115 and 130 mg/L, respectively. P. aeruginosa 78 and 99 strains emulsified benzene and n-hexane at the highest rates, and the surfaces of these strains were 73% and 65% and 62% and 72% more hydrophobic for benzene and toluene, respectively.