Assessing the prevalence of airborne fungi in a city or region is critical to diagnosing the ecology and effectively treating allergic manifestations induced by inhaling fungal allergens. The present study was conducted to evaluate the characteristics and variations of culturable airborne fungi at four selected sampling sites in Hangzhou in southeastern China. Results showed that the concentration of culturable fungi in the air ranged from < 12 colony forming units CFU m–3 to 8767 CFU m–3 with a mean of 848 CFU m–3. We identified a total of 352 fungal isolates from multiple sampling sites and across seasons, which were distributed among 21 genera and 85 species of fungi. Penicillium, Cladosporium, Alternaria, Aspergillus, and Trichoderma were the most predominant fungi, based on frequency and concentration, and the fungal composition differed both between sites and between seasons. Approximately 36.5% of the total number of isolated fungal species belonged to Penicillium, which also contributed the largest proportion of the total fungal concentration (about 29.6%). The most prevalent fungal species in Hangzhou were P. chrysogenum (7.7%), C. cladosporioides (6.3%), Alternaria alternata (5.6%), P. funiculosum (4.3%), and Aspergillus sydowii (4.0%). Moreover, significant variation existed between the sampling sites, with the highest fungal concentrations detected on Zhejiang Gongshang University Jiaogong Campus (ZJGSUJC) and Breeze-ruffled Lotus at Quyuan Garden (BLQG), followed by those on Yan’an Road Business Street (YRBS). The lowest concentrations (P < 0.05) were found on Tianmushan and Jiaogong Cross Road (TJCR). Furthermore, the concentrations at the four sites exhibited different seasonal patterns, with the lowest mean concentrations occurring during winter at all of the locations, whereas no difference was detected between summer, autumn, and spring. Our results provide a baseline for studying culturable airborne fungi in southeastern China, thus allowing the health risks from exposure to the atmosphere to be evaluated.