Exposure to bioaerosols has been associated with respiratory allergies and asthma. This highlights a need to characterize the fungal flora along an urbanization gradient. In this study, the airborne fungal flora, the concentration of dominant fungi and their seasonal variation along an urbanization gradient were investigated through viable count and direct microscopic count. Thirty-one fungal species including Cladosporium, Aspergillus, Penicillium, Fusarium and Trichoderma were identified. Characteristics of fungal floras differed significantly among seasons. The lowest spore concentration presented in winter, constituting the baseline data of airborne fungi. Urban and rural areas had significantly different airborne fungal floras and spore concentration. Aspergillus flavus and C. oxysporum were the dominant fungi in summer and autumn, respectively. Spatial distributions of dominant fungi, A. flavus and C. oxysporum, in their dominant seasons were similar and in both cases their spore concentration increased along a gradient of vegetation coverage. This pattern seems to be a result of the relatively lower plant tissue and soil in the urban. Contrasting with the dominance of allergenic C. herbarum in temperate areas, the dominant fungus in tropical Taiwan was C. oxysporum.