Microbiological indicators (total coliforms, faecal coliforms, E. coli and enterococci) and phylogenetic groups (domain Archaea, classes Alpha-, Beta-, Gammaproteobacteria and the Cytophaga-Flavobacterium group) detected by FISH were detected in profiles of a highly anthropogenically-affected stream (Luzická Nisa; Northern Bohemia, Czech Republic). This study aimed to assess the changes in the microbial communities of such a polluted stream, and possible relationships between “classic indicators” and the phylogenetic groups. One particular aim concerned a characterisation of the fluorescence in situ hybridization (FISH) method, the source of any uncertainty and its limit in terms of quality control (QA/QC). Of the phylogenetic groups studied, the Proteobacteria phylum was more abundant in comparison to the Cytophaga-Flavobacterium group or the Archaea domain. The profile Lucany (above the start of city urban areas) was very different from later downstream profiles, because of its very low faecal bacteria content, low counts of Gammaproteobacteria, and evident dominance of the Cytophaga-Flavobacterium group together with Betaproteobacteria. Later profiles did not show such large differences among themselves. The group of Gammaproteobacteria was very common mainly in profiles with high amounts of untreated faecal pollution. The repeatability of counting bacteria by the FISH method was 14 % on average, an “uncertainty” similar to that of cultivation methods.