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Background Invasive fungal infections (IFIs) represent a significant threat to immunocompromised individuals, particularly patients with hematologic malignancies such as acute myeloid leukemia (AML). Intensive chemotherapy and prolonged neutropenia place these patients at high risk for opportunistic fungal pathogens, including Aspergillus spp. and Candida spp. IFIs not only increase hospitalization duration and healthcare costs, but are also associated with poor long-term survival outcomes. Current antifungals face major limitations: increasing rates of resistance, and substantial toxicity profiles. Chimeric Antigen Receptor (CAR) T-cell therapy, has shown promise beyond oncology. We have demonstrated in preclinical models that CAR T-cells engineered to recognize Aspergillus-specific antigens can significantly reduce fungal burden in murine models of pulmonary aspergillosis. The next critical step is to develop an off-the-shelf, bioengineered CAR T-cell product that can be administered to patients with suspected or confirmed IFIs.Methods We designed a new AF-CAR, based on the Fab domain of the Aspergillus fumigatus-specific monoclonal antibody AF-549. The AF-CAR extracellular domain was constructed by fusing the single-chain variable fragment (scFv) derived from AF-549 with the extracellular portion of the human Fc domain, enhancing stability and potential effector interaction. The co-stimulatory domain was from CD28, and the T-cell activation domain was from CD3ζ, domain. AF-CAR T cells and AF-CAR natural killer T (NKT) cells—were generated using a third-generation lentiviral vector system. Fungicidal activity was evaluated through microscopic analysis of hyphal damage. Additional cytokine profiling was conducted to quantify effector responses.Results AF-CAR T cells demonstrated robust and specific recognition of Aspergillus fumigatus (strain Af293) conidia and hyphae in co-culture assays. Fluorescence microscopy confirmed targeted interaction of AF-CAR T cells with both hyphal tips and mature hyphal filaments, indicating effective fungal engagement. Functionally, AF-CAR T cells significantly inhibited fungal growth across all cytokine conditions. After 18 hours of co-culture, AF-CAR T cells achieved a 55–62% reduction in hyphal length at a 100:1 effector-to-target (E:T) ratio relative to control cultures. Similar expansion profile was observed with AF-CAR iNKT cells. Fluorescence microscopic analysis confirms AF-NKT cells targeting and killing AF-hyphae.Conclusions We developed a novel strategy for generating lentivirally transduced AF-CAR T cells and AF-CAR iNKT cells with high targeting specificity and potent antifungal activity against Aspergillus fumigatus in vitro. The next critical phase of development involves validating AF-CAR iNKT therapy in relevant in vivo models to comprehensively assess antifungal efficacy, biodistribution, safety, and potential off-target effects.