AbstractCopper (Cu)-based fungicides have been used in viticulture to prevent downy mildew since the end of the 19th century, and are still used today to reduce fungal diseases. Consequently, Cu has built up in many vineyard soils, and it is still unclear how this affects soil functioning. The present study aimed to assess the short and medium-term effects of Cu contamination on the soil fungal community. Two contrasting agricultural soils, an acidic sandy loam and an alkaline silt loam, were used for an eco-toxicological greenhouse pot experiment. The soils were spiked with a Cu-based fungicide in seven concentrations (0–5000 mg Cu kg⁻¹soil) and alfalfa was grown in the pots for 3 months. Sampling was conducted at the beginning and at the end of the study period to test Cu toxicity effects on total microbial biomass, basal respiration and enzyme activities. Fungal abundance was analysed by ergosterol at both samplings, and for the second sampling, fungal community structure was evaluated via ITS amplicon sequences. Soil microbial biomass C as well as microbial respiration rate decreased with increasing Cu concentrations, with EC₅₀ranging from 76 to 187 mg EDTA-extractable Cu kg⁻¹soil. Oxidative enzymes showed a trend of increasing activity at the first sampling, but a decline in peroxidase activity was observed for the second sampling. We found remarkable Cu-induced changes in fungal community abundance (EC₅₀ranging from 9.2 to 94 mg EDTA-extractable Cu kg⁻¹soil) and composition, but not in diversity. A large number of diverse fungi were able to thrive under elevated Cu concentrations, though within the order ofHypocrealesseveral species declined. A remarkable Cu-induced change in the community composition was found, which depended on the soil properties and, hence, on Cu availability.