Cake layer formation on the membrane surface has been a major challenge in the operation of membrane bioreactors (MBRs). In this study, the cake layer formation mechanism in an MBR used for synthetic wastewater treatment was investigated. The major components of cake layer were systematically examined by particle size analyzer (PSA), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray fluorescence (XRF), energy-diffusive X-ray analyzer (EDX), and Fourier transform infrared (FTIR) spectroscopy. The results indicate that the small particles in sludge suspension had a strong deposit tendency on the membrane surface. The SEM and CLSM analysis exhibited that bacterial clusters and polysaccharides were significant contributors to membrane fouling. The main components of biopolymers were identified as proteins and polysaccharide materials by the FTIR. The examination by EDX and XRF demonstrated that Mg, Al, Ca, Si, and Fe were the major inorganic elements in fouling cake. Furthermore, the results suggest that bridging between deposited biopolymers and inorganic compounds could enhance the compactness of fouling layer. During the operation of MBRs, the biopolymers and inorganic elements in the bioreactor should be controlled to minimize membrane fouling.