Dibenzothiophene (DBT) is a typical recalcitrant thiophenic sulfur component of fuels, and its desulphurization has been a model reaction in the treatment of these compounds. Based on this information, the potential of Pseudomonas fluorescens (UCP 1514) on the desulfurization of dibenzothiphene was studied, in order to use it for reducing the sulfur content of diesel oil in compliance with environmental regulations. The result of biodegradation by the bacteria was determined by undertaking high-performance liquid chromatography of the metabolites produced. These can also be identified by gas chromatography with a mass spectrometry detector, and doing so revealed a sulfur-free product, biphenyl, as the final product of the degradation process. The results showed a decrease of 73% in dibenzothiophene content, which means that P. fluorescens removes sulfur from dibenzothiophene with a good selectivity to form biphenyl. These promising results indicate that P. fluorescens has an interesting potential to degrade sulfur-containing compounds in diesel oil and thereby could help in removing sulfur content from diesel oil. The process of microbial desulfurization described herein can be used particularly after carrying out hydrodesulfurization. Consequently, the sulfur content could be reduced even further. Applying P. fluorescens UCP 1514 in dibenzothiophene could help to understand the nature of the biodegradation process and to achieve the regulatory standards for sulfur level in fossil fuels.