Bacterial communities associated with a variety of benthic detritus types were studied in three streams in the context of the chemical characteristics of the sediment material and the stream water. A cell purification assay was developed for a quantitative microscopic evaluation of bacterial community structure in detritus samples by fluorescence in situ hybridization (FISH). The efficiency of FISH with fluorescently monolabelled probes was compared with FISH with signal amplification by catalysed reporter deposition (CARD-FISH). In detritus types poor in organic carbon and nitrogen, the numbers of prokaryotes were related to the chemical characteristics of the stream water column, whereas no such relationship was found for detritus types rich in organic carbon and nitrogen. These results might help to provide criteria for the selection of detritus types for river ecosystem assessment and monitoring. The percentage of bacteria detected by FISH with monolabelled probes was correlated with the detritus total organic matter (OM). This is likely attributed to a higher ribosome content of microbial cells on substrates rich in OM. Cell detection by CARD-FISH did not show any correlation with OM content, indicating that this technique renders the results more independent from the activity state of cells. Fluorescence in situ hybridization with four group-specific probes suggested a relationship between substrate quality and the composition of the microbial assemblages on the various types of detritus. The improved protocol for cell purification and CARD-FISH may facilitate future investigations on the relationship between the riverine benthic detritus quality and microbial community composition.