The purpose of this study was to document and characterize the dynamic formation of biofouling using confocal scanning laser microscopy (CSLM) and to investigate the roles microbial aggregates and their associated extracellular polymeric substances (EPS) have on biofouling development. The effect of sludge retention time (SRT) on biofouling development and mixed liquor properties was also examined. Three pilot scale MBRs were used in this study operating at three different SRTs (6, 10 and 15 days). Under steady state conditions within the range of SRTs tested, an increase in SRT related to an in EPS concentration. The results confirmed an increase in cTOC relates to an increase in sEPS. As the protein: carbohydrate (P/C) ratio increased the zeta potential became less negative. A clear relationship was found between biofouling resistance and biofilm thickness as well as oxygen uptake rate suggesting biofilm growth is the most important mechanism in membrane fouling. The biofilm thickness increased as the SRT increased. The development of biofilm on membrane surface was initiated by the attachment mainly from proteins. In contrast, continual accumulation and compaction of carbohydrates became increasingly more important, particularly for biofilm layer away from the membrane surface.