Petroleum products which are used in a wide variety of industries as energy sources and raw materials have become a major concern in pollution of terrestrial and marine environments. The purpose of this study was to assess the potential of indigenous microbial isolates for degradation of diesel fuel. Two most proficient bacterial strains among five isolated strains from polluted soil of an industrial refinery were studied. The isolates then were identified as Pseudomonas aeruginosa and Bacillus subtilis using biochemical tests and 16S rRNA gene sequence analyses. P. aeruginosa showed higher biodegradation efficiency than B. subtilis in shaking flask containing diesel-contaminated water. P. aeruginosa and B. subtilis degraded about 87 and 75% of total hydrocarbons, respectively, in flasks containing 2% diesel and 98% water. The biodegradation efficiency of the isolates decreased as diesel contamination increased from 2 to 5%. The isolates showed significantly higher efficiency on degradation of short-chain hydrocarbons in 20 days, i.e., by using P. aeruginosa, removal efficiency of C-10 hydrocarbons was near 90%, while about 69% of C20+ hydrocarbons and 47% of aromatic hydrocarbons were removed. Therefore, the isolates showed high capability in biodegradation of diesel contamination of the refinery.