Various attempts have been made to develop biotechnological processes based on microbiological desulfurization employing aerobic and anaerobic bacteria. In order to obtain a biodesulfurization process competitive with the chemical-physical one of hydrodesulfurization (HDS), a biotechnological process has to follow the three main refinery steps: 1) separation, entailing some pretreatment of crude oil; 2) conversion, i.e. biocatalytic transformation where the biocatalyst favours a selective desulfurization process without destroying useful products; 3) finishing, in which the crude oil is separated from the biocatalyst and the byproducts. Biocatalysis may be carried out using whole cells or isolated enzymes in the free or immobilized form. The use of isolated enzymes is advantageous since it avoids the formation of undesirable byproduct mediated by contaminating enzymes as in the case of the aerobic biodesulfurization, which is not a selective process. In fact, the aerobic microorganisms may degrade almost all the compounds which make up the heavy oil. However, despite this advantage, extraction and purification of the enzyme is costly and, frequently, enzymes catalyzing oxidation-reduction reactions require enzyme cofactors which must be regenerated after the reaction. For these reasons, metabolic conditions can often be designed by using whole cell biotransformations to promote cofactor regeneration, thus avoiding the problems associated with cofactor recycling and regeneration. Today, the main limitations for the industrial application of a biodesulfurization process are associated with the high cost of the biocatalyst and with the volumetric ratio between the organic phase and the aqueous one. In order to overcome these problems, cell immobilization is one of the most promising approaches in terms of treatment costs and in finishing step times when compared to a continuous stirred tank bioreactor for the biodesulfurization process.