Aim 1. To disentangle the effects of local environmental conditions and space at multiple scales in order to uncover and explain biogeographical signals in microbial communities of freshwater diatoms. 2. To assess the role of species traits related to dispersal capacities in diatom biogeography. Location Rivers and streams of Sweden sampled over a total area of 450,000 km2. Methods We used partial redundancy analyses and variation partitioning coupled with spatial eigenfunctions analyses to separate the effects of environment and space at three different scales on diatoms community structure. As community analyses might give limited insight into species‐specific mechanisms, we also performed species by species variation partitioning and the amount of variance explained by each fraction was related to species cell length and attachment to the substrate. Results We found a strong effect of environment and space at a large scale suggesting environmental filtering and dispersal limitation over large distances. Additionally, species‐based analyses uncovered an effect of space at fine scale suggesting possible species‐specific mass effect dynamic or species sorting driven by missing environmental factors varying at fine scale. Environmental and spatial effects varied strongly among species, but this variation was not explained by cell length and attachment to the substrate. Main conclusions Our results confirm the importance of both environment and dispersal‐related processes in controlling diatom community structure and bring new perspectives on the role of dispersal in shaping microbial communities at biogeographical scale. Dispersal being a species‐specific mechanism, species associations are not necessarily conserved from site to site and species‐based approaches can detect different spatial patterns than those derived from traditional community analyses. Contrary to the conclusions of small‐scale analysis of connected metacommunities, interspecific trait variation seems to be of limited importance for long‐distance overland dispersal in diatoms.