Membrane biofouling by microbial products adversely impacts the feasibility of adopting membrane bioreactors (MBRs) for treating wastewater. The fouling layer structure determines the pressure drop across the fouling layer. Three-dimensional distributions of nucleic acids, proteins, alpha-D-GLUCOPYRANOSE POLYSACCHARIDES, AND B-D-glucopyranose polysaccharides in the fouling layer formed on a mixed cellulose ester membrane were generated utilizing a quadruple staining protocol combined with confocal laser scanning microscopy (CLSM). For the first time, this study constructed a three-dimensional volumetric grids model representing the fouling layer structure on the basis of a series of CLSM images. Quantitative structural information about the fouling layer was extracted from the CLSM images.