Aim: To determine the microbial composition of biofilms in domestic toilets by molecular means. Methods and Results: Genomic DNA was extracted from six biofilm samples originating from households around Dusseldorf, Germany. While no archaeal 16S rRNA or fungal ITS genes were detected by PCR, fingerprinting of bacterial 16S rRNA genes revealed a diverse community in all samples. These communities also differed considerably between the six biofilms. Using the Ribosomal Database Project (RDP) classifier tool, 275 cloned 16S rRNA gene sequences were assigned to 11 bacterial phyla and 104 bacterial genera. Only 15 genera (representing 121 sequences affiliated with Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes and Proteobacteria) occurred in at least half of the samples or contributed at least 10% of the sequences in a single biofilm. These sequences were defined as 'typical' for toilet biofilms, and they were examined in more detail. On a 97% sequence similarity level, these sequences represented 56 species. Twelve of these were closely related to well-described bacterial species, and only two of them were categorized as belonging to risk group 2. No 16S rRNA genes of typical faecal bacteria were detected in any sample. Virtually all 'typical' clones were found to be closely related to bacteria or to sequences obtained from environmental sources, implicating that the flushing water is the main source of recruitment. Conclusion: In view of the great diversity of mostly yet-uncultured bacteria and the considerable differences between individual toilets, very general strategies appear to be most suited for the removal and prevention of toilet biofilms. Significance and Impact of the Study: For the first time, a molecular fingerprinting and cloning approach was used to monitor the species composition in biofilm samples taken from domestic toilets. Knowledge about the microbial composition of biofilms in domestic toilets is a prerequisite for developing and evaluating strategies for their removal and prevention.