Microbial growth develops in stored diesel fuel. This can influence later use in emergency backup generators and vehicles through the clogging of fuel filters by the development of biomass. A mutualistic microbial community can develop in stored fuel products that degrade polycyclic aromatic hydrocarbons to the benefit of one another. This project aimed to study the bacterial community profile of diesel obtained from commercial fuel stations using culture dependent and independent methods. Bacteria and total DNA were isolated from the diesel and the community was studied using denaturing gradient gel electrophoresis of the 16S rDNA region. This resulted in the isolation of six bacterial species from two different genera (related to Bacillus and Lysinibacillus) that could grow in pure culture with diesel as carbon source. The denaturing gradient gel electrophoresis analysis indicated the occurrence of an additional eight genera in the sampled diesel. Both methods identified bacteria related to Bacillus pumilus occurring in the samples. Bacillus pumilus was the predominant species (50%) isolated from diesel samples using culture media. The identification of bacteria occurring in commercial diesel samples can be useful in determining the degree of degradation occurring during the storage of the product and also possible bioremediation agents in diesel fuels spills.