Water and wastewater treatment solutions incorporating biofilm systems are becoming increasingly popular due to more stringent regulations pertaining to drinking water and wastewater effluent discharge in Canada and in other parts of the world. As a major component of biofilm, extracellular polymeric substances (EPS) have been considered as an important factor affecting the physical and chemical properties of biofilm. Further, the selected method of EPS extraction and the methods of detecting the composition of the EPS have shown to affect the results of EPS measurements. In this research, protocols for EPS extraction and EPS composition analysis were investigated and optimized for nitrifying moving bed biofilm reactor (MBBR) biofilm. In addition, the confocal Raman microscopy (CRM) spectra of EPS in nitrifying MBBR biofilm and the protein, polysaccharide and extracellular DNA (eDNA) percent concentrations of the EPS were investigated at various operating temperatures. Further, the CRM spectra and the protein, polysaccharide and eDNA percent concentration of EPS in nitrifying MBBR biofilm along with the biofilm morphology and thickness and the viability of the embedded cells were investigated at various hydraulic retention times (HRTs). The EPS was characterized at various temperatures and HRTs in order to investigate potential correlation between the EPS components of the nitrifying biofilm and the ammonia removal kinetics. The biofilm morphology and thickness along with the bacterial viability of the biofilm were also investigated at various HRTs. Biofilm morphology images and thickness measurements were acquired using a variable pressure scanning electron microscope (VPSEM). The percentages of viable embedded cells in the biofilm were quantified using live/dead staining in combination with confocal laser microscopy (CLSM) imaging. The research demonstrates that an increase in protein content and subsequently a decrease in polysaccharides and eDNA contents in the EPS of nitrifying MBBR biofilm were observed at the lowest operational HRT and the highest temperature in this work. In particular, the EPS protein to polysaccharide (PN/PS) ratio of nitrifying MBBR systems was shown to significantly decrease below a value of 3 when the system was underloaded (observed at the highest operational temperature in this study) or hydraulically overloaded (observed at the lowest HRT in this study). As such, data obtained at lower operational temperatures, with the system no longer underloaded, and at longer HRTs, with the system no longer hydraulically overloaded, all demonstrate an EPS PN/PS ratio of approximately 3. Correlations were observed between the chemically measured EPS PN/PS ratios and the measured Raman spectra intensity ratios; supporting the concept of higher PN/PS ratios of EPS in more optimal nitrifying MBBR operations. Further, the ammonia removal kinetics and EPS response at HRT values of 0.75 and 1.0 h indicate that nitrifying MBBR systems may be optimized to operate at HRTs as low as 0.75 to 1.0 hour as opposed to conventional HRTs of 2.0 to 6.0 h.