Phosphorus (P) acquisition is key for plant growth. Arbuscular mycorrhizal fungi (AMF) help plants acquire P from soil. Understanding which factors drive AMF-supported nutrient uptake is essential to develop more sustainable agroecosystems. Here we collected soils from 150 cereal fields and 60 non-cropped grassland sites across a 3,000 km trans-European gradient. In a greenhouse experiment, we tested the ability of AMF in these soils to forage for the radioisotope 33P from a hyphal compartment. AMF communities in grassland soils were much more efficient in acquiring 33P and transferred 64% more 33P to plants compared with AMF in cropland soils. Fungicide application best explained hyphal 33P transfer in cropland soils. The use of fungicides and subsequent decline in AMF richness in croplands reduced 33P uptake by 43%. Our results suggest that land-use intensity and fungicide use are major deterrents to the functioning and natural nutrient uptake capacity of AMF in agroecosystems.