The identification of collected airborne unicellular fungal conidia and hyphae using nonviable techniques is subjective and an imprecise process. Similarly, to determine whether an individual is allergic to a particular genus requires a separate immunodiagnostic analysis. This study demonstrates the development of a novel double immunostaining halogen assay, which enables (1) the simultaneous identification of collected airborne fungal conidia and hyphae of Aspergillus fumigatus and Penicillium chrysogenum using monoclonal antibodies and (2) the demonstration of patient-specific allergy to the same particles using human serum IgE. The results demonstrate that when conidia were ungerminated the binding of antibodies was homogeneous and localized in close proximity around the entire conidia for both species. However, when conidia were germinated, the proportion expressing antigen increased (P < 0.0001) for both species and the sites of binding of the two antibodies changed with double immunostaining restricted to the hyphal tips for A. fumigatus, in addition to the sites of germination for P. chrysogenum. The described immunoassay has the potential to identify fungal particles in personal environmental air samples, provided species-specific monoclonal antibodies are available, while simultaneously demonstrating allergic sensitization to the same particles by co-staining the samples with the patient's own serum. Such an immunoassay can use those fungi that the patient is actually exposed to and potentially avoids many problems associated with extract variability based on the performance of current diagnostic techniques for fungal allergy.