Biofilm formation on synthetic membranes, i.e., membrane biofouling, is a major problem encountered in membrane processes for water and wastewater treatment. Quantification of biofouling is often conducted destructively and hence, results only reflect a snapshot of biofouling processes. This limitation is mainly due to the lack of tools that allow us to monitor dynamics of biofouling without the need of dissembling the membrane testing systems. In a recent study, we developed a novel multichannel fluidic membrane biofilm flow cell that enables non-destructive monitoring of biofouling dynamics using confocal laser scanning microscopy (CLSM). As a proof of concept, we used Green Fluorescent Protein (GFP)-tagged Shewanella oneidensis as a model organism and examined its biofilm development on forward osmosis membranes. The temporal profiles of quantitative biofouling parameters were obtained without disrupting the continuous operation of the membrane testing system. We also demonstrated that, combining with fluorescent staining techniques; the dynamics of biofouling by natural, un-tagged bacteria could also be monitored using CLSM without dissecting the membranes. The microfluidic flow cell developed in this study is a promising tool for non-destructive evaluation of antifouling properties of novel membranes.