In biotechnology, composition of biofilms and suspended bioaggregates can be crucial for system performance or product quality. Consequently, understanding biofilm dynamics is important for any process optimisation. The aim of this study was to investigate biofilm development and detachment under different hydrodynamic conditions and varying glucose load. Confocal laser scanning microscopy proved to be a fast method providing information about structure, distribution and volume ratio of bacteria and extra cellular polymers (EPS) within biofilms and detached biomass. As a result, it could be shown that biofilm structure, in terms of density and EPS volume, was largely influenced by hydrodynamic conditions. Furthermore, it was demonstrated that the EPS:bacteria ratio and distribution was largely influenced by substrate load. Finally, the characteristics in biofilm structure and development were reflected in the composition and quantity of the detached biomass.