This study was conducted to evaluate the hydrocarbon biodegradation abilities of Enterobacter cloacae, Staphylococcus aureus, Sphingomonas paucimobilis, and Pentoae species which were isolated from different diesel-contaminated soil samples. The isolates were identified by the Vitek 2 system. Fourier-transform spectroscopy (FT-IR) tested the potential of these isolates to biodegrade the diesel according to the peak areas, a significant decrease in the area of the peaks at 2856-2928 cm−1 corresponds to aliphatic hydrocarbons. The appearance of small peaks at 900-1032 cm−1 refers to substituted benzene derivative compounds. An appearance of some new peaks at 3010- 3030 cm−1 which indicate the presence of alcohol (-OH) and ketones (RC=O). A sharp peak appeared at 1712 cm−1 refers to the carbonyl group (C=O). The potential of biological surfactant production was tested using the Sigma 703D stand-alone tensiometer showed that these isolates were biological surfactant producers. The better results of the surface tension reduction test were obtained when using the mixed bacterial culture which reduced the surface tension of the medium from 66 mN/m to 35.15 mN/m. Single isolates and mixed bacterial culture have investigated their ability to degrade 3.0 % (v/v) of diesel as sole source of carbon and energy in Bushnell- Haas medium. The results demonstrated that the bacterial isolates could be effective in biodegradation of diesel spills individually and showed good biodegradation abilities when they are used together in the mixed bacterial culture.