The effects of biodiversity of aboveground organisms have been widely investigated in a range of ecosystems, yet whether similar responses are also seen in belowground microbial communities, such as ectomycorrhizal (EM) fungi, are little understood. We investigated, in vitro, the effects of a gradient of 1–8 species of EM fungi interacting with substratum carbon:nitrogen (C:N) ratio on biomass production and CO2 efflux. The model experimental systems enabled us to recover and measure biomass of individuals within communities and calculate net selection and complementarity effects. Both biomass and CO2 efflux increased with species richness particularly under high N concentrations. Moreover, net biodiversity effects were largely positive, driven by both selection and complementarity effects. Our results reveal, in pure culture, the implications of EM species richness on community productivity and C cycling, particularly under high N conditions, and constitute the basis for future experiments under natural conditions.