In this study, biomass characteristics including bacterial community, extracellular polymeric substances (EPS) production, and membrane fouling propensity were examined when the membrane bioreactors (MBRs) were fed with different substrates (i.e., different C/N/P ratios). Denaturing gradient gel electrophoresis (DGGE) analysis revealed that significant shifts of bacterial communities happened when increasing nitrogen or phosphorus loading in the MBRs, which followed in an almost similar way. At steady state, the biomass from the low C/N- and C/P-MBRs had comparable concentrations and produced similar EPS levels as those in the control MBR. However, the median particle size increased when the MBRs fed with low C/N- or C/P-substrate, possibly associated with the filamentous bacteria propagating in the MBRs. Increasing nitrogen or phosphorus loading 1-fold could not induce more serious membrane fouling compared to the control MBR.