The aim of our study was to identify the dominant environmental niche (pipe wall and different sediment size fractions) in the distribution system for biological activity, Aeromonas and the occurrence of several microbial groups or specific opportunistic pathogens. The pipe wall and sediment fractions of three locations in a distribution system were sampled and different microbiological parameters were determined. The results demonstrated that most of the active biomass in the distribution system is located in the pipe wall biofilm and the smallest sediment size fraction. Fungi were more dominantly present in the pipe wall biofilm than in the sediment, whereas Aeromonas spp. has its dominant niche in the sediment. The highest numbers of the opportunistic pathogens Pseudomonas aeruginosa and Aspergillus fumigatus were observed in the biofilm. In contrast, Stenotrophomonas maltophilia was only observed in the sediment. The results from our study demonstrate that: (i) microbial activity in the distribution system is high on the pipe wall and sediment and (ii) different microbial groups or microbial species have different dominant environmental niches in the distribution system.