The biodegradation of No. 2 diesel fuel under anaerobic conditions was investigated using sediments collected from wetlands of Barataria-Terrebonne estuary in Louisiana. The results indicated enhanced biodegradation of diesel fuel under sulfate-reducing, nitrate-reducing, methanogenic, and mixed electron acceptor conditions. However, the rate of diesel degradation was the highest under mixed electron acceptor conditions followed in order by sulfate-reducing, methanogenic, and nitrate-reducing conditions. Under mixed electron acceptor condition, 99% removal of diesel fuel was achieved within 510 days, while under sulfate-reducing condition 62% degradation of diesel fuel was observed for the same period. Diesel fuel was also degraded to a smaller extent in the culture condition where electron acceptors were not supplemented (natural attenuation condition). This study showed evidence for enhanced diesel fuel metabolism in a mixed microbial population system similar to any contaminated field site, where a heterogeneous microbial population exists.