Anaerobic degradation of petroleum hydrocarbon is very relevant in removing oil spills in the anaerobic zones. of soil and sediments. This research investigated the possibility of degrading petroleum hydrocarbon under sulfate condition. Diesel fuels were degraded significantly under sulfate reducing condition within 182 days and 65% degradation of petroleum hydrocarbon was observed in the reactor operation. This research showed evidence for diesel fuel metabolism in a mixed microbial population system similar to any contaminated field sites, where heterogeneous microbial population exists.