The purpose of this study was to characterize microbial floc structure and properties under phosphorus (P) limiting and non-limiting regimes. The P-limitation applied to the biomass did not significantly impact on reactor performance in terms of COD removal and MLSS. The composition of EPS was affected by the P-limitation with significantly increased accumulation of carbohydrates, uronic acids and proteins. CLSM and glycoconjugate mapping revealed that the relative abundance of α and β- N -acetylgalactosaminyl/galactopyranosyl and N -acetylglucosaminyl residues was affected by P-limitation, suggesting changes in microbial populations within the floc structure, which in turn could cause the compositional changes of EPS. The image analysis performed on CLSM images indicated that under non-limiting conditions the cell clumps within the floc were significantly smaller as compared to P-limiting conditions. The fractal dimension and porosity under limiting conditions were either significantly higher or lower than under P-rich conditions.