The procedure currently used to diagnose infection in otitis externa has several limitations: it is slow to culture organisms on growth media, fungal infections are often missed, and extensive laboratory facilities and mycological expertise are required. A rapid, accurate and sensitive assay would greatly improve patient care by initiating appropriate antifungal treatment at the onset of disease. We report the development of a rapid detection assay for otomycosis using fungal-specific monoclonal antibodies to detect fungi in ear swabs by immunofluorescence microscopy. This assay could form the basis of a detection assay for fungal infections of the head and neck.