The efficacy Biological wastewater treatment process is largely dependent on the formation of microbial flocs and settleability before the water is released into the environment. Settleability and flocculation are reliant upon stable physicochemical parameters. Extracellular polymeric substance constituents dictate physicochemical parameters of flocs. The fluctuation of these constituents within mixed microbial flocs is poorly studied. A novel aspect of this research was the use of CLSM data to get a semi- quantitative assessment of the constituents within mixed microbial flocs. Wastewater treatment flocs were characterized for eubacterial ecology, physicochemical properties, and they were visualized through correlative microscopy. It was observed that the microbial communities from the three sampling sites exhibited significant variability in numerous physicochemical properties. Overall, these results provide a first step to examine micro-localization of physicochemical properties, architecture and processes within flocs that may help better understand the causes of floc- related inefficiencies in biological wastewater treatment.