The structure of the Bacteria and Archaea community in a large drinking water reservoir (Marathonas, Greece; MR) was investigated in October 2007 and September 2008, using 16S rRNA gene clone libraries. The bacterial communities were more diverse than archaeal communities (Shannon diversity index H′ 0.81–3.28 and 1.36–1.77, respectively). The overall bacterial community composition was comparable to bacterioplankton community described in other freshwater habitats. Within the Bacteria, Betaproteobacteria dominated, while representatives of Alpha-, Gamma- and Deltaproteobacteria also occurred. Other important phyla were Actinobacteria and Bacteroidetes, while representatives of Acidobacteria, Cyanobacteria, Chloroflexi, Planctomycetes and Verrucomicrobia were also retrieved. Several phylotypes in Alpha- and Betaproteobacteria and Bacteroidetes were related to bacteria capable of cyanotoxin degradation and with aromatic compounds/iron oxidizers or polymer degraders. Euryarchaeota dominated (60.5%) the Archaea community mostly with phylotypes related to Methanobacteriales and Methanosarcinales. Among the Thaumarchaeota, the two most abundant phylotypes were affiliated (97% similarity) with the only cultivated mesophilic thaumarchaeote of marine origin, Nitrosopumilus maritimus. Temporal and spatial comparison of the prokaryotic community structure revealed that three of the most abundant prokaryotic phylotypes, belonging to Actinobacteria, were recovered from all sites both years, suggesting that these Actinobacteria could be important key players in MR ecosystem functioning.