Invasive fungal infections are a major cause of infection related mortality in immunocompromised patients. Furthermore, inappropriate immune responses to fungal pathogens are associated with disease exacerbations in particular in patients with allergies or compromised lung functions, such as asthma or cystic fibrosis. Antigen-specific CD4+ T cells have been suggested to play a major role in anti-fungal immune responses. However, the exact quantification and detailed characterization of fungus-specific CD4+ T cells in humans has been hampered so far by the lack of appropriate methods for their identification. To overcome this technology gap, a flow-cytometric method was developed that allows for the first time the direct ex vivo detection and comprehensive characterization of the entire fungus-reactive CD4+ T cell compartment from human peripheral blood, i.e. conventional memory and naive CD4+ T cells (Tcon), as well as regulatory T cells (Treg). Based on the magnetic pre-enrichment of fungal antigen stimulated CD154+ Tcon and CD137+ Treg, fungus-reactive CD4+ T cells can be analyzed from large starting cell numbers, which allows the collection of sufficient numbers of target cells for subsequent phenotypic and functional analyses with high statistical precision. This technology was used to perform a detailed characterization of the CD4+ T cell response against two of the major fungal pathogens in humans, the constantly inhaled airborne fungus Aspergillus fumigatus and the gut- and skin-residing commensal Candida albicans.