The filtration membranes utilized in advanced treatment of municipal wastewater are prone to biological fouling. Permeability loss and water recovery limitations due to fouling necessitate additional pretreatment and membrane cleaning, which raise the cost of water reuse. Better fouling mitigation calls for a deeper understanding of biofilm behavior on water reuse membranes. This study uses confocal microscopy to monitor biofouling under realistic conditions. To simulate water reuse, a miniature flow cell with microfiltration and ultrafiltration membranes was operated under typical filtration conditions using secondary effluent from California’s East Bay Municipal Utility District. The growing biofilms were stained for extracellular polymeric substances and live and dead cells, and they were monitored in situ under filtration conditions with a confocal laser scanning microscope to produce 2-D and 3-D images. In situ image series revealed biofilm growth and removal processes including internal and external fouling, extracellular polymeric substance production, wrinkling, delamination, and film re-deposition. Additionally, changes in biofilm morphology between in situ and ex situ images highlighted limitations of ex situ imaging. These results provide insight into the physical and biological mechanisms of biofouling in hopes of informing the development of improved techniques for biofouling mitigation.