Diverse microorganisms form complex microbial communities and usually exist in biofilm communities in both natural environments and engineered systems such as a wastewater treatment process. However, the conventional approach to investigate microbial ecology has not contributed to the understanding and clarification of the structure and function of biofilm communities. Some effective methods have been developed to investigate phylogenetic affiliations, metabolic activities and genetic activities in biofilm communities at the single-cell level. These techniques have been contributing to a better understanding of the spatial organization of biofilm communities and activities in engineered systems. However, further effort is needed to set out the general rules governing community development in biofilm communities and to advance the process performance of engineered systems. This review describes advances and limitations in methodology, particularly focusing on fluorescence in situ hybridization (FISH) and related techniques and the application of these methods to nitrifying biofilms in wastewater treatment processes.