The soluble microbial products (SMP) and extracellular polymeric substances (EPS) significantly affect fouling in membrane filtration. This work filtered SMP and fractionated EPS of activated sludge using a 0.45mm filter membrane. The fluorescently labeled stains and confocol laser scanning microscopy (CLSM) images in situ visualized the distributions of proteins, -polysaccharides, -polysaccharides, lipids, total cells, and dead cells of different EPS fractions in the fouling layers. The role of individual SMP and fractionated EPS fractions on membrane fouling was discussed.