Biotransformation of soybean biodiesel and the inhibitory effect of petrodiesel were studied under methanogenic conditions. Biodiesel removal efficiency of more than 95% was achieved in a chemostat with influent biodiesel concentrations up to 2.45 g/L. The kinetics of anaerobic biodegradation of soybean biodiesel B100 (biodiesel only) with different petrodiesel loads was studied using biomass pre-acclimated to B100 and B80 (80% biodiesel and 20% petrodiesel). The results indicated that the biodiesel fraction of the blend could be effectively biodegraded, whereas petrodiesel was not biodegraded at all under methanogenic conditions. The presence of petrodiesel in blends with biodiesel had a greater inhibitory effect on the rate of biodegradation than the biodegradation efficiency (defined as the efficiency of methane production). Both the biodegradation rate coefficient and the methane production efficiency increased almost linearly with the increasing fraction of biodiesel. With the increasing fraction of petrodiesel, the biodegradation rate and efficiency were correlated with the concentration of soluble FAMEs in the water.