In this study, 65 yeast strains were isolated from different environmental samples contaminated with various petroleum hydrocarbons such as activated sludges and soil samples from automobile workshops. The yeast isolates were tested for biosurfactant production using various screening methods such as parafilm M test, oil displacement assay, drop collapse assay, determination of surface tension reduction, and emulsification index. Nineteen of the isolates were found positive for biosurfactant production and their molecular characterizations were carried out by sequencing analysis of the ITS1-5.8S-ITS2 region and D1/D2 domain of 26S rDNA. The results indicated that these strains were from a wide range of yeast genera including Rhodotorula, Candida, Yarrowia, Geotrichum, Galactomyces, and Cystobasidium. The studies to determine the emulsification index revealed that the biosurfactants produced by Yarrowia lipolytica strains (TEMGS33, TEMOS12, and TEMOS14) and Apiotrichum loubieri strain (TEMOS16) were the most potent and capable of forming stable emulsions with emulsion index (E-24) up to 68%. In addition, quantitative measurements of the surface tension reduction of the biosurfactants produced by these strains were carried out by Du Nouy ring method. Biosurfactants produced from Yarrowia lipolytica strain TEMGS33 and Apiotrichum loubieri strain TEMOS16 gave the best results reducing the surface tension to 34.7 +/- 1.15 and 35.3 +/- 0.55mNm(-1), respectively. Based on these data, biosurfactants from Yarrawia lipolytica strains (TEMGS33, TEMOS12, and TEMOS14) and Apiotrichum loubieri strain (TEMOS16) showed promising results and might be implemented in numerous industrial fields such as bioremediation and food industry.