We present a comprehensive dataset on the bacterioplankton diversity along a 2 600 km midstream transect of the second largest river in Europe, the Danube River, and its tributaries using an Illumina-based sequencing approach (16S rRNA-gene amplicon sequencing). Our analysis revealed a clear gradual development of the riverine bacterioplankton community in both the 0.2-3.0 µm and the > 3.0 µm size fractions along the entire river. We found that bacterial richness and evenness gradually decreased downwards the river.