Streams and rivers form conspicuous networks on the Earth and are among nature's most effective integrators. Their dendritic structure reaches into the terrestrial landscape and accumulates water and sediment en route from abundant headwater streams to a single river mouth. The prevailing view over the last decades has been that biological diversity also accumulates downstream. Here, we show that this pattern does not hold for fluvial biofilms, which are the dominant mode of microbial life in streams and rivers and which fulfil critical ecosystem functions therein. Using 454 pyrosequencing on benthic biofilms from 114 streams, we found that microbial diversity decreased from headwaters downstream and especially at confluences. We suggest that the local environment and biotic interactions may modify the influence of metacommunity connectivity on local biofilm biodiversity throughout the network. In addition, there was a high degree of variability in species composition among headwater streams that could not be explained by geographical distance between catchments. This suggests that the dendritic nature of fluvial networks constrains the distributional patterns of microbial diversity similar to that of animals. Our observations highlight the contributions that headwaters make in the maintenance of microbial biodiversity in fluvial networks.