Introduction and objective: Detection of the common allergen airborne fungi in any region is critical for prevention and treatment of allergic fungal diseases. Therefore, this study was conducted to identify the most common airborne fungal species of Isfahan, investigate their allergic reactions in animal and obtain local fungal strains for use as antigens in allergy tests to be used.Materials and methods: An open plate method was used to scan airborne fungal contents over 12 months in Isfahan. On the same days every week, triplicate samples were collected at three different locations in the morning, at noon and in the evening. The fungal culture media were incubated at 25oC until growth appeared and then the airborne fungi were identified by routine mycological laboratory methods. The extracts of the most common airborne fungi isolated, were examined with skin prick test for allergic reactions in laboratory animals. Results: During this study, the most abundant airborne fungi identified in Isfahan were species of yeasts, yeast like (Candida spp., Geotrichum spp., and Trichosporon spp.), and mold (Cladosporium spp., Penicillium spp., Aspergillus spp., and Alternaria). Positive skin reactions were observed with Cladosporium spp.(40%), and Aspergillus spp. (21%), Alternaria spp. (17%), Penicillum spp. (15%) and Geotrichum spp. (7%). Conclusion: It is concluded that fungi have a significant role in infecting immunocompromised hosts, information obtained in the present study contribute toward a better understanding of the pattern of occurrence of airborne fungi, and may assist allergists, clinicians and epidemiologists to treat the diseases.