We analyzed the bacterial 16S rRNA gene diversity throughout the major components of the drinking water distribution system of a ca. 52,000-inhabitants city (Trikala City, Greece) in order to describe the changes of the bacterial assemblages and to detect possible bacterial pathogens which are not included in the standard monitoring process. Bacterial DAPI counts and DNA extraction was performed in the water pumping wells, the water treatment tank and tap water from households. Approximately 920 bp of the bacterial 16S rDNA were PCR-amplified, cloned, and sequenced for a total of 191 clones, which belonged to 112 unique phylotypes. The water of the pumping wells harbored a typical subsurface bacterial assemblage, with no human pathogens, dominated by beta-Proteobacteria. Cell abundance in the water treatment tank decreased significantly, close to detection limit, but bacterial diversity remained high. However, the dominance of beta-Proteobacteria decreased considerably, indicating the sensitivity of this group to drinking water disinfection treatment. Tap water from the households hosted a much less diverse, low-cell bacterial assemblage, dominated by Mycobacterium-like phylotypes, related to biofilm bacterial communities.