The current understanding of membrane fouling in submerged membrane bioreactors is still insufficient. Therefore, the role of microbial polymeric substances in membrane fouling mechanisms in a submerged membrane bioreactor was systematically investigated in this study. Microbial polymeric foulants accumulated on the membrane surface and in its pores. The foulants were then extracted in three steps, without deforming the membrane structure. Moreover, filtration resistances were determined by the resistance in series model. A strong functional relationship was observed between total polymeric substances accumulated in the membrane and their fouling resistances. The greatest accumulation of polymeric substances occurred in cake layers formed on the membrane surface. Correspondingly, the greatest fouling resistance resulted from the cake layer. The results indicated that more protein accumulated on the membrane surface than carbohydrates because the proteins were stickier. Regarding the analysis of microbial polymeric substances, it was observed that the filtration with a 0.45 mu m pore size filter did not capture all of the polymeric substances that took part in membrane fouling.