Microbial biogeography studies expend much effort in determining whether environmental selection or stochastic processes related to dispersal are more important in shaping community composition. While both types of factors are possibly influential, it is tacitly assumed that protists, or microbial eukaryotes in general, behave biogeographically as prokaryotes because of their small physical size. However, direct evidence for this in exactly the same environment and at the same phylogenetic depth is lacking. In this study, we compared the structure of both prokaryotic and eukaryotic components of microbial communities forming biofilms on mineral substrates in different geographic locations at the level of small-subunit (SSU) rRNA-based operational taxonomic units (OTUs). These microbial communities are subjected to strong environmental selection and contain significant proportions of extremophilic microorganisms adapted to desiccation and UV radiation. We find that the nature of the substrate as well as climatic variables and geography influences microbial community structure. However, constrained correspondence analyses and distance–decay curves showed that, whereas the substrate type was the most significant factor structuring bacterial communities, geographic location was the most influential factor for microbial eukaryote communities. Biological explanations implying a higher dispersal success for bacteria combined with more mobile lifestyles for predatory protists may underlie these different prokaryote versus microbial eukaryote biogeographic patterns.