The influence of biofilm structure on transport and transformation processes in biofilms has been investigated microscopically using microelectrodes, a micro-slicing procedure and various chemical and microbiological tests. The study demonstrates that the biofilm structure is highly stratified, characterized by an increase of biofilm density, a decrease of metabolically active biomass, and a decrease of porosity with biofilm depth. Both the effective diffusivity for dissolved oxygen and the effectiveness factor decrease with biofilm depth. Competition for substrate and space in biofilms results in this stratified structure, which is also affected by biofilm thickness. The study reveals that there are different trends for the density increase and the decreases of porosity, microbial activity and DO effective diffusivity with biofilm depth for different biofilm thicknesses. The results of this study are helpful in obtaining a clearer physical description of biofilms, and help to bridge the gap between the mathematical modelling and external-phenomenon observation of biofilm systems.