Phototrophic biofilms may be defined as interfacial microbial communities mainly driven by light as energy source. Structure, productivity and taxonomic composition of freshwater phototrophic biofilms under different growth conditions were investigated within the EU-project PHOBIA with the following aims: 1) optimisation of wastewater treatment in wetlands, 2) control and prevention of biofouling on submersed objects, and 3) modelling of phototrophic biofilm development. Experiments were carried out in a flow-lane incubator with precise control of external light, temperature, velocity conditions and nutrient-adapted artificial medium. Structure and architecture of phototrophic biofilms at different developmental stages were examined by using multi-channel contocal laser scanning microscopy (CLSM). The development of phototrophic biofilms was clearly light dependent. Fast growing phototrophic biofilms were mostly dominated by single species algae and formed less stable structures of up to 900 μm thickness. Biofilms with these dimensions had to be cryo-sectioned and post-stained for CLSM. Laser microscopy analysis also revealed a stratification of phototrophic organisms which was more pronounced in slow growing biofilms. In contrast, at very low light intensity the development of phototrophic biofilms was strongly delayed. In conclusion, structural features and subsequent functional relationships may be key parameters for exploitation, control and modelling of phototrophic biofilms.