For an efficient operation in biodesulfurization of petroleum and related fuels, the aqueous solubility of insoluble or very slightly soluble sulfur compounds contained in the petroleum products has to be increased. In this study, polyoxyethylene nonionic surfactants were used in order to enhance the aqueous solubility of insoluble or very slightly soluble sulfur compounds contained in the bunker-C oil and the solubilized sulfur contents in the aqueous surfactant solutions were measured by X-ray sulfur spectrophotometer. The most hydrophobic surfactant used during this study showed the maximum solubilization capacity for sulfur compounds contained in the bunker-C oil and the solubilization of sulfur compounds was found to increase with temperature and to be abruptly increased at above 5 wt % surfactant concentrations. It was found that Tergitol series surfactants showed higher solubilizing capacity than Neodol series surfactants presumably due to the disruption of the regular packing in the hydrocarbon region of the surfactant micellar aggregates and that the addition of a cosurfactant and/or an electrolyte increased the solubilization of sulfur compounds in the bunker-C oil. It was also shown that partitioning phenomena were shown to be significant with a hydrophobic surfactant especially at high temperature and pH of the Tergitol surfactant solution did not affect the solubilization of sulfur compounds. The growth of M6 sulfur-reducing bacteria was not greatly affected by the addition of both nonionic surfactant and cosurfactant. Desulfurization experiments with M6 sulfur-reducing bacteria showed that the biodesulfurization rate of bunker-C oil was enhanced with addition of nonionic surfactant and these data suggested the potential applicability of surfactant to the actual biodesulfurization system.