Copper-based fungicides have been used for a long time in viticulture and have accumulated in many vineyard soils. In this study, incrementing Cu(OH) 2-based fungicide application from 0.05 to 5 g Cu kg −1 on two agricultural soils (an acidic sandy loam (L, pH 4.95) and an alkaline silt loam (D, pH 7.45)) resulted in 5 times more mobile Cu in the acidic soil. The most sensitive parameters of alfalfa (Medicago sativa) growing in these soils were the root nodule number, decreasing to 34% and 15% of the control at 0.1 g Cu kg −1 in soil L and at 1.5 g Cu kg −1 in soil D, respectively, as well as the nodule biomass, decreasing to 25% and 27% at 0.5 g Cu kg −1 in soil L and at 1.5 g Cu kg −1 in soil D, respectively. However, the enzymatic N 2-fixation was not directly affected by Cu in spite of the presence of Cu in the meristem and the zone of effective N 2-fixation, as illustrated by chemical imaging. The strongly different responses observed in the two tested soils reflect the higher buffering capacity of the alkaline silt loam and showed that Cu mitigation and remediation strategies should especially target vineyards with acidic, sandy soils.