The aim of this work was to optimize the anaerobic treatment of a waste stream from an enhanced oil recovery (EOR) process. The treatment of a simulated waste water containing about 150 mg chemical oxygen demand (COD)/L of total petroleum hydrocarbons (TPH) and the saturation level of CO(2) was evaluated. A two-step anaerobic system was undertaken in the mesophilic temperature range (30-40 degrees C). The method of evolutionary operation EVOP factorial design was used to optimize pH, temperature and organic loading rate with the target parameters of CO(2) reduction and CH(4) production in the first reactor and TPH removal in the second reactor. The results showed 98% methanogenic removal of CO(2) and CH(4) yield of 0.38 L/gCOD in the first reactor and 83% TPH removal in the second reactor. In addition to enhancing CO(2) and TPH removal and CH(4) production, application of this method showed the degree of importance of the operational variables and their interactive effects for the two reactors in series. (C) 2010 Elsevier Ltd. All rights reserved.