The contribution of human neutrophils to the protection against fungal infections by Aspergillus fumigatus is essential but not fully understood. Whereas healthy people can inhale spores of A. fumigatus without developing disease, neutropenic patients and those receiving immunosuppressive drugs have a higher incidence of invasive fungal infections. To study the role of neutrophils in protection against A. fumigatus infections, we developed an in vitro assay in which the interactions between human neutrophils and A. fumigatus were observed in real time, at single-cell resolution, in precisely controlled conditions. We measured the outcomes of neutrophil-fungus interactions and found that human neutrophils have a limited ability to migrate toward A. fumigatus and block the growth of A. fumigatus conidia (proportion with growth blocked, 69%). The blocking ability of human neutrophils increased to 85.1% when they were stimulated by uniform concentrations of fMLP and was enhanced further, to 99.4%, in the presence of chemoattractant gradients. Neutrophils from patients receiving immunosuppressive treatment after transplantation were less effective against the fungus than those from healthy donors, and broader heterogeneity exists between patients, compared with healthy individuals. Further studies using this microfluidic platform will help understand the relevance of innate immune deficiencies responsible for the higher risk of fungal infections in patients with immunosuppressive disease.