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Abstract Aim Climate change and anthropogenic environmental deterioration strongly affect aquatic microbial communities. Although microbes have irreplaceable roles in various ecosystems, the spatial variation in microbial communities has received less attention in comparison to macro‐organisms. Studies aiming to disentangle the effects of local environmental, catchment and climatic variables on microbial communities are also rare. Here, we disentangled the effects of local, catchment, spatial and climatic variables on boreal stream diatom and bacterial communities. Location Western Finland Methods We sampled 21 boreal river basins comprising 105 study sites spanning 520 km in north‐south direction and 330 km in east‐west direction in western Finland. We used principal coordinates of neighbour matrix analysis ( PCNM ), redundancy analyses ( RDA ), variation partitioning, boosted regression trees ( BRT ) and regression analyses to examine variation in community composition and species richness. Results Water chemistry and physical variables had significant effects on the community composition of both microbial groups. Catchment level variables explained a slightly larger amount of variation in diatom community composition than local level variables. Agriculture was the most significant determinant of variation in diatom community composition among catchment level variables and was also related with variation in the richness of both groups. Of spatial and climatic variables, growing degree days ( GDD ) and spatial variables were the most significant drivers determining diatom community composition. GDD was also positively associated with the richness of diatoms and bacteria. Unique effects of spatial and climate variables accounted for the largest amount of variation in the community composition of both diatoms and bacteria. Main conclusions Our results highlight that aquatic microbial communities can exhibit biogeographical variation at regional scales due to the joint influence of local, catchment and climatic variables, and possibly because of dispersal limitation. Catchment properties, especially agriculture, can be used as a proxy for the effects of landscape alteration on aquatic microbial communities.