The replacement of native birch with Norway spruce has been initiated in Norway to increase long-term carbon storage in forests. However, there is limited knowledge on the impacts that aboveground changes will have on the belowground microbiota. We examined which effects a tree species shift from birch to spruce stands has on belowground microbial communities, soil fungal biomass and relationships with vegetation biomass and soil organic carbon (SOC). Replacement of birch with spruce negatively influenced soil bacterial and fungal richness and strongly altered microbial community composition in the forest floor layer, most strikingly for fungi. Tree species-mediated variation in soil properties was a major factor explaining variation in bacterial communities. For fungi, both soil chemistry and understorey vegetation were important community structuring factors, particularly for ectomycorrhizal fungi. The relative abundance of ectomycorrhizal fungi and the ectomycorrhizal : saprotrophic fungal ratio were higher in spruce compared to birch stands, particularly in the deeper mineral soil layers, and vice versa for saprotrophs. The positive relationship between ergosterol (fungal biomass) and SOC stock in the forest floor layer suggests higher carbon sequestration potential in spruce forest soil, alternatively, that the larger carbon stock leads to an increase in soil fungal biomass.