The phylogenetic composition, bacterial biomass, and biovolume of both planktonic and biofilm communities were studied in a low-order Bystřice stream near Olomouc City, in the Czech Republic. The aim of the study was to compare the microbial communities colonizing different biofilm substrata (stream aggregates, stream sediment, underwater tree roots, stream stones, and aquatic macrophytes) to those of free-living bacteria. The phylogenetic composition was analyzed using fluorescence in situ hybridization for main phylogenetic groups. All phylogenetic groups studied were detected in all sample types. The stream stone was the substratum where nearly all phylogenetic groups were the most abundant, while the lowest proportion to the DAPI-stained cells was found for free-living bacteria. The probe specific for the domain Bacteria detected 20.6 to 45.8 % of DAPI-stained cells while the probe specific for the domain Archaea detected 4.3 to 17.9 %. The most abundant group of Proteobacteria was Alphaproteobacteria with a mean of 14.2 %, and the least abundant was Betaproteobacteria with a mean of 11.4 %. The average value of the Cytophaga- Flavobacteria group was 10.5 %. Total cell numbers and bacterial biomass were highest in sediment and root biofilm. The value of cell biovolume was highest in stone biofilm and lowest in sediment. Overall, this study revealed relevant differences in phylogenetic composition, bacterial biomass, and biovolume between different stream biofilms and free-living bacteria. [ABSTRACT FROM AUTHOR]