The nature of the cake formed on a membrane surface is a key factor for understanding membrane fouling phenomenon in membrane bioreactor (MBR) systems. Most studies on membrane fouling have been carried out with the analysis of microbial characteristics in the bulk phase rather than in the cake because the preparation of intact cake specimen is hardly possible. In this study, the cake on a membrane surface in a MBR system was non-destructively examined using confocal laser scanning microscopy (CLSM) and digital image analysis. This CLSM-Image analysis technique was then applied to elucidate the mechanism of significant flux enhancement that occurs when a membrane fouling reducer (MFR) was added to MBR. Even though the cake of the MFR reactor was thicker than that of the control reactor, membrane fouling was decreased primarily as the result of increased cake porosity. The concentration of soluble extra-cellular polymeric substances (EPS) in the MFR reactor was lower, but the bound EPS concentration was higher than that in the control reactor. Cakes formed in the presence vis-a-vis at the absence of MFR were compared in terms of structural features and the spatial distribution of cellular and polymeric cake constituents which were determined using the CLSM data and image analysis. (c) 2006 Elsevier B.V. All rights reserved.