Functionally, ectomycorrhizal (ECM) and saprotrophic (SAP) fungi belong to different guilds, and they play contrasting roles in forest ecosystem C-cycling. SAP fungi acquire C by degrading the soil organic material, which precipitates massive CO₂ release, whereas, as plant symbionts, ECM fungi receive C from plants representing a channel of recently assimilated C to the soil. In this study, we aim to measure the amounts and identify the drivers of ECM and SAP fungal biomass in temperate forest topsoil. To this end, we measured ECM and SAP fungal biomass in mineral topsoils (0–12 cm depth) of different forest types (pure European beech, pure conifers, and mixed European beech with other broadleaf trees or conifers) in a range of about 800 km across Germany; moreover, we conducted multi-model inference analyses using variables for forest and vegetation, nutritive resources from soil and roots, and soil conditions as potential drivers of fungal biomass. Total fungal biomass ranged from 2.4 ± 0.3 mg g⁻¹ (soil dry weight) in pure European beech to 5.2 ± 0.8 mg g⁻¹ in pure conifer forests. Forest type, particularly the conifer presence, had a strong effect on SAP biomass, which ranged from a mean value of 1.5 ± 0.1 mg g⁻¹ in broadleaf to 3.3 ± 0.6 mg g⁻¹ in conifer forests. The European beech forests had the lowest ECM fungal biomass (1.1 ± 0.3 mg g⁻¹), but in mixtures with other broadleaf species, ECM biomass had the highest value (2.3 ± 0.2 mg g⁻¹) among other forest types. Resources from soil and roots such as N and C concentrations or C:N ratios were the most influential variables for both SAP and ECM biomass. Furthermore, SAP biomass were driven by factors related to forest structure and vegetation, whereas ECM biomass was mainly influenced by factors related to soil conditions, such as soil temperature, moisture, and pH. Our results show that we need to consider a complex of factors differentially affecting biomass of soil fungal functional groups and highlight the potential of forest management to control forest C-storage and the consequences of changes in soil fungal biomass.