Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted invariant natural killer T (iNKT) cells compose a small subset of T cells known to impact the immune response toward various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined by CD69 expression and cytokine production in response to distinct fungal morphotypes in the presence of different CD1d+ antigen presenting cells using flow cytometry and multiplex enzyme-linked immunosorbent assay (ELISA). Among CD1d+ subpopulations, CD1d+CD1c+ mDCs showed the highest potential to activate iNKT cells on a per cell basis. The presence of A. fumigatus decreased this effect of CD1d+CD1c+ mDCs on iNKT cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1 and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating function for human iNKT cells during A. fumigatus infection.