The soluble (S), loosely bound (LB) and tightly bound (TB) extracellular polymeric substances (EPS) were extracted from sludge flocs of a membrane bioreactor to evaluate their characteristics and adsorptive fouling. The degrees of adsorptive fouling by the EPS fractions were in the order S-EPS < TB-EPS < LB-EPS. The images of atomic force microscopy showed the membrane fouled by LB-EPS was rougher than that fouled by the other fractions. The adsorbed EPS layer, which was sensed by quartz crystal microbalance with dissipation, was found to be more rigid and compact for LB-EPS, compared with the other EPS fractions. The excitation–emission matrix and Fourier transform infrared techniques were also used to characterize the individual EPS fractions. Compared with S-EPS and TB-EPS, the LB-EPS contained a larger amount of aromatic protein and less carbohydrates and lipids, exhibiting characteristics of greater aromaticity and hydrophobicity. These characteristics should be responsible for more severe fouling, and the stiffer and more compact structure of the adsorbed layer.