Biofilm development on the membrane surface is one of the main reasons for membrane fouling in membrane bioreactors (MBRs) and it is a big problem for their stable operation. Precise information on the microbial community composition of the biofilm is needed for a better understanding of biofilm development. However, there have been limited investigations of the relationship between the biofilm formation process and the microbial community of activated sludge and biofilm in MBRs treating real sewage. In this study, relationships between the microbial community structure of biofilm and activated sludge at each biofilm formation stage were investigated and biofilm growth was elucidated by nondestructive observations. Two anoxic/oxic MBRs were operated and membrane fouling was induced. Permeability rapidly decreased in both reactors and live cell microcolonies were formed on dead cell conditioning film on the membrane surface. Principal component analysis based on 16S rRNA gene sequences showed that the biofilm microbial community changed significantly from middle stage to mature biofilm when compared with that of activated sludge. The abundance of specific bacteria, such as unclassified Neisseriaceae, increased in middle-stage biofilm and the diversity indexes of middle-stage biofilm were lower than those of mature biofilm and activated sludge. These results suggested that the presence of specific bacteria with colonization ability played a crucial role in biofilm formation. Strategies are needed to target membrane fouling mitigation during early- and middle-stage biofilm formation to reduce MBR membrane fouling.