The human-pathogenic fungus Aspergillus fumigatus can cause life-threatening infections, particularly in immunosuppressed or neutropenic patients with hematological malignancies. The human antifungal immune response primarily relies on neutrophils with contribution from macrophages, while the roles of unconventional lymphocytes such as Natural Killer (NK) cells and mucosal-associated invariant T (MAIT) cells remain less understood. Therefore, using an advanced microfluidic “invasive aspergillosis-on-chip” (IAC) model, we investigated the coordinated antifungal response of various immune cell types in combination with macrophages within a human tissue-like 3D environment. The IAC model, initially incorporating monocyte-derived macrophages, was enhanced by perfusing either primary neutrophils, NK cells, or MAIT cells for overnight infection experiments. In this model hyphal growth of A. fumigatus was only prevented when both macrophages and neutrophils were present. However, uncontrolled growth was detected in neutropenic patient scenarios. We observed key interactions such as conidial shuttling and metaforosis between innate immune cells, accompanied by elevated cytokine levels of e.g. IL-1β and TNFα. Importantly, NK cells applied to the vascular compartment of the IAC model significantly reduced hyphal length, confirming their antifungal activity. Similarly, MAIT cells in sufficiently high quantities reduced hyphal growth and branching, although this effect depended on the recognition of riboflavin-derived antigens, as shown by the lack of response to the A. fumigatus ΔriboB mutant strain. Together, our findings demonstrate that the advanced IAC model is a powerful tool to dissect human antifungal immunity. The interactions observed between macrophages and other immune cells highlight the importance of multi-layered defense strategies against A. fumigatus.