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The bacterioplankton diversity in large rivers has thus far been undersampled, despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along a midstream transect of the Danube River and its tributaries. Using 16S rRNA-gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free-living and particle-associated bacterial communities. These shifts were also supported by the beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwater and typical of lakes, whereas only few taxa attributed to lotic systems were detected. These freshwater taxa, which were composed of members of the acI lineage and the freshwater SAR11 group (LD12) and the Polynucleobacter, increased in proportion downriver and were accompanied by a decrease in soil and groundwater bacteria. When examining our results in a broader ecological context, we elaborate that patterns of bacterioplankton diversity in large rivers can be explained by the River Continuum Concept published in 1980, with a modification for planktonic microorganisms. (version 2.0 submitted to Environmental Microbiology 2014-Sept-09)