Small-scale hydraulics affects microbial behaviour at the cell level1, trophic interactions in marine aggregates2 and the physical structure and function of stream biofilms3,4. However, it remains unclear how hydraulics, predictably changing from small streams to large rivers, impacts the structure and biodiversity of complex microbial communities in these ecosystems. Here, we present experimental evidence unveiling hydraulics as a hitherto poorly recognized control of microbial lifestyle differentiation in fluvial ecosystems. Exposing planktonic source communities from stream and floodplain ecosystems to different hydraulic environments revealed strong selective hydraulic pressures but only minor founder effects on the differentiation of attached biofilms and suspended aggregates and their biodiversity dynamics. Key taxa with a coherent phylogenetic underpinning drove this differentiation. Only a few resident and phylogenetically related taxa formed the backbone of biofilm communities, whereas numerous resident taxa characterized aggregate communities. Our findings unveil fundamental differences between biofilms and aggregates and build the basis for a mechanistic understanding of how hydraulics drives the distribution of microbial diversity along the fluvial continuum5,​6,​7.