A continuous bench-scale submerged membrane bioreactor (BSMBR) was operated in two modes; low (LC) and optimum calcium (OC) concentrations, to investigate the effect of calcium on membrane biofouling. Both the cake layer and pore blocking resistances were reduced in the operation under OC conditions. In order to find the causes of fouling, molecular weight fractionations of the extracellular polymeric substances (EPS) and soluble microbial products (SMP) were conducted, and the relative hydrophobicity of the sludge, EPS and SMP were also measured. The reduction in the cake layer resistance was assumed to be due to the decrease of filamentous bacteria, and better flocculation caused by the calcium bridges and the increased hydrophobicity of EPS in the operation under OC conditions. It was suggested that the pore blocking resistance was reduced due to the low SMP rejection of the membrane and the fewer hydrophobic reactions of the SMP in the operation under OC conditions. Fourier-transform infrared analysis of the fouled membrane surface supported that proteins and carbohydrates were the main foulants during the BSMBR operation.