The performance of a stirred tank bioreactor operated in sequencing batch operation mode (SBR) to treat diesel waste from storage tanks, was investigated. The SBR run in four cycles of 72 h, using optimized setpoints for pH, initial waste load, C:N ratio and agitation speed. Optimal conditions allowed the system to reach biodegradation percentages of 53.3, 96.0, 76.2 and 75.0% at the end of cycles one, two three and four, respectively. Investigations of microbial diversity showed changes in the microbial community members at the end of the cycle one. Significant reductions in the relative ecotoxicity were observed beginning with cycle two, and the reductions extended until the end of process. The SBR operation mode proved to be an efficient method for treating the diesel waste, allowing relevant reductions in the hydrocarbon content of the waste along with an increase in its environmental quality. © 2010 Elsevier Ltd.