Aim Ecoregions and the distance decay in community similarity are fundamental concepts in biogeography and conservation biology that are well supported across plants and animals, but not fungi. Here we test the relevance of these concepts for ectomycorrhizal (ECM) fungi in temperate and boreal regions. Location Europe. Time Period 2008–2015. Major Taxa Studied Ectomycorrhizal fungi. Methods We used a large dataset of ~24,000 ectomycorrhizas, assigned to 1350 operational taxonomic units, collected from 129 forest plots via a standardized protocol. We investigated the relevance of ecoregion delimitations for ECM fungi through complementary methodological approaches based on distance decay models, multivariate analyses and indicator species analyses. We then evaluated the effects of host tree and climate on the observed biogeographical distributions. Results Ecoregions predict large-scale ECM fungal biodiversity patterns. This is partly explained by climate differences between ecoregions but independent from host tree distribution. Basidiomycetes in the orders Russulales and Atheliales and producing epigeous fruiting bodies, with potentially short-distance dispersal, show the best agreement with ecoregion boundaries. Host tree distribution and fungal abundance (as opposed to presence/absence only) are important to uncover biogeographical patterns in mycorrhizas. Main Conclusions Ecoregions are useful units to investigate eco-evolutionary processes in mycorrhizal fungal communities and for conservation decision-making that includes fungi.