Confocal laser scanning microscopy (CLSM) combined with fluorescent viability indicators, was used in this study to investigate the impact of hospital wastewaters on floc structure and composition. In this work, three pilot-scale projects, two membrane bioreactors (MBRs) with a submerged or external membrane bioreactor and a conventional activated sludge, were installed and operated for 65 days. They were fed with an influent sampled directly from the hospital drainage system, which contained micropollutant concentrations ranging from ng/L to mg/L. Samples of flocs were observed using CLSM to characterize the extracellular polymeric substances (EPS) stained with concanavalin A–tetra methylrhodamine and fluorescein isothiocyanate solution and combined with a fluorescent viability indicator (Baclight® Bacterial Viability Kit, Molecular Probes), allowing visualization of isolated stained cells in the three-dimensional structure of flocs (damaged or not). The results of CLSM of the sludge composition were compared with classical biochemical analysis of EPS made through a thermal extraction method. The results showed a good relation between these analyses and the statistical treatment of microscopic pictures.