Biofilms are layered structures of microbial cells and an extracellular matrix of polymeric substances, associated with surfaces and interfaces. Biofilms trap nutrients for growth of the enclosed microbial community and help prevent detachment of cells from surfaces in flowing systems. Phototrophic biofilms can best be defined as surface attached microbial communities mainly driven by light as the energy source with a photosynthesizing component clearly present. Eukaryotic algae and cyanobacteria generate energy and reduce carbon dioxide, providing organic substrates and oxygen. The photosynthetic activity fuels processes and conversions in the total biofilm community, including the heterotrophic fraction. This thesis starts with a brief introduction in the ecology of phototrophic biofilms and discusses their actual and potential applications in wastewater treatment, bioremediation, fish-feed production, biohydrogen production, and soil improvement and their role in biofouling. The next chapter describes the diversity of phototrophic bacteria in hot spring microbial mats found on the east coast of Greenland. In this study we utilized a polyphasic approach using a combination of isolation techniques, microscopic observation of morphological features, and cultivation-independent molecular methods. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different microbial mat parts. Chapter 4 focuses on the successional changes in community composition of freshwater phototrophic biofilms growing under different light intensities. Our results suggest that surface colonization by heterotrophic pioneers facilitates the development of phototrophic biofilms. In Chapter 5 we compared the community composition of phototrophic biofilms cultivated in three microcosm systems operated under identical conditions but placed in different laboratories. Denaturing Gradient Gel Electrophoresis (DGGE) analysis of both 16S and 18S rRNA gene fragments showed that the communities developed differently in terms of species richness and community composition. Chapter 6 demonstrates that nifD gene sequences, coding for a nitrogenase subunit, can be used to detect and identify diazotrophic cyanobacteria in natural communities. PCR products generated using primers homologous to conserved regions in the cyanobacterial nifD genes were subjected to DGGE and clone library analysis in order to determine the genetic diversity of diazotrophic cyanobacteria in environmental samples. In the last chapter we describe the development of PCR primers targeting conserved regions within the cyanobacterial hupS gene family. This gene is involved in the hydrogen metabolism of diazotrophic microorganisms. We analyzed hupS diversity and transcription in cultivated phototrophic biofilms by the direct retrieval and analysis of mRNA that was reverse transcribed, amplified with hupS specific primers, and cloned. Overall, the community composition and species richness of phototrophic biofilms was shown to be highly variable. Cultivation-independent molecular methods proved very useful to study diversity and function in phototrophic biofilms.