Despite many modern wastewater treatment solutions, the most common is still the use of activated sludge (AS). Studies indicate that the microbial composition of AS is most often influenced by the raw sewage composition (especially influent ammonia), biological oxygen demand, the level of dissolved oxygen, technological solutions, as well as the temperature of wastewater related to seasonality. The available literature mainly refers to the relationship between AS parameters or the technology used and the composition of microorganisms in AS. However, there is a lack of data on the groups of microorganisms leaching into water bodies whose presence is a signal for possible changes in treatment technology. Moreover, sludge flocs in the outflow contain less extracellular substance (EPS) which interferes microbial identification. The novelty of this article concerns the identification and quantification of microorganisms in the AS and in the outflow by fluorescence in situ hybridization (FISH) method from two full-scale wastewater treatment plants (WWTPs) in terms of 4 key groups of microorganisms involved in the wastewater treatment process in the context of their potential technological usefulness. The results of the study showed that Nitrospirae, Chloroflexi andCa. Accumulibacter phosphatis in treated wastewater reflect the trend in abundance of these bacteria in activated sludge. Increased abundance of betaproteobacterial ammonia-oxidizing bacteria and Nitrospirae in the outflow were observed in winter. Principal component analysis (PCA) showed that loadings obtained from abundance of bacteria in the outflow made larger contributions to the variance in the PC1 factorial axis, than loadings obtained from abundance of bacteria from activated sludge. PCA confirmed the reasonableness of conducting studies not only in the activated sludge, but also in the outflow to find correlations between technological problems and qualitative and quantitative changes in the outflow microorganisms.