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• Contents of soil bacterial and fungal biomass were quantified across Europe. • Land use and climate jointly control soil bacterial and fungal biomass. • Soil agricultural use buffers the impact of harsh climate conditions on bacteria. • Soil texture, organic carbon and nitrogen are predictors of soil microbial biomass. • Soil microbial biomass was not influenced by crop type in croplands. Elucidating contents and drivers of soil bacterial and fungal biomass in contrasting land uses and climates at European scale is useful to define appropriate policies for the conservation of the ecosystem services that soil microorganisms provide. Here, we aimed to (i) quantify and compare bacterial and fungal biomass in 513 European soils collected from three different land uses (forests, grasslands, and croplands) and climates (arid, temperate, and cold) through analysis of fatty acid methyl esters; (ii) model the factors controlling soil bacterial and fungal biomass; and (iii) investigating levels of bacterial and fungal biomass in cropland soils cultivated with three important crop types in Europe: cereals, oil-producing crops, and orchards. Bacterial biomass decreased with land use in the following order: grasslands > croplands > forests and was found to be the highest in temperate environments. Similar patterns were found for biomass of Gram-positive and Gram-negative bacteria and Actinobacteria. Soil fungal biomass was greater in forests than in croplands and grasslands and was favoured by colder environments. The fungi to bacteria ratio (F/B) decreased as follows: forests > croplands > grasslands, with soils in colder climates showing greater F/B ratios in croplands and forests. Soil texture, soil organic carbon, and nitrogen were shown to directly drive bacterial and fungal biomass. The biomass of the different microbial groups was not influenced by the crop type when only croplands were considered. Fungi appear to be more susceptible to agricultural soil use than bacteria. Moreover, agricultural use of soil seems to buffer the effect of harsh climatic conditions on soil bacterial biomass. The present study improves our understanding of the combined effects of land use and climate on soil bacterial and fungal biomass across Europe. [ABSTRACT FROM AUTHOR]