River waters are classified worldwide in relation to their chemical quality status, which is assessed by the presence of inorganic and organic pollutants. Meanwhile, microbiological quality evaluations are based mainly on the occurrence of pathogens and faecal contaminants, which are indirectly retrieved by standard cultivation methods. In this study, the structural dynamics of a riverine bacterioplankton community were investigated through a quantitative single-cell approach with the use of flow cytometry for the quantification of total, viable and active cells. Furthermore, catalyzed reporter deposition (CARD)-FISH was used for the evaluation of Proteobacteria and Cytophaga–Flavobacteria bacterial classes. The aim was to identify the major cytometric and phylogenetic cell groups that most representatively reflect the physical and chemical changes occurring in an upland-to-lowland transect along a human-impacted river (the Foglia River, Italy). The use of CARD-FISH to obtain cell abundance measurements of specific taxonomic clusters enabled different microbial propagation dynamics along the river transects to be identified. Furthermore, our results highlighted that the multiparametric approach with flow cytometry produces basic microbiological parameters in a reasonable time span that is consistent with the requirements for an early warning monitoring strategy with respect to integrated riverine water quality assessments. Copyright © 2015 John Wiley & Sons, Ltd.