AbstractTransplant recipients on calcineurin inhibitors are at high risk of invasive fungal infection. Understanding how calcineurin inhibitors impair fungal immunity is a key priority for defining risk of infection. Here, we show that the calcineurin inhibitor tacrolimus impairs clearance of the major mould pathogenAspergillus fumigatusfrom the airway, by inhibiting macrophage inflammatory responses. This leads to defective early neutrophil recruitment and fungal clearance. We confirm these findings in zebrafish, showing an evolutionarily conserved role for calcineurin signalling in neutrophil recruitment during inflammation. We find that calcineurin–NFATactivation is phagocytosis dependent and collaborates withNF‐κB forTNF‐α production. For yeast zymosan particles, activation of macrophage calcineurin–NFAToccurs via the phagocytic Dectin‐1–spleen tyrosine kinase pathway, but forA. fumigatus, activation occurs via a phagosomalTLR9‐dependent and Bruton's tyrosine kinase‐dependent signalling pathway that is independent of MyD88. We confirm the collaboration betweenNFATandNF‐κB forTNF‐α production in primary alveolar macrophages. These observations identify inhibition of a newly discovered macrophageTLR9–BTK–calcineurin–NFATsignalling pathway as a key immune defect that leads to organ transplant‐related invasive aspergillosis.