During the one-year period of this study, 288 samples were taken and 954 fungal colonies were isolated from the air of Isfahan, Iran. Among the fungi isolated, Cladosporium, yeasts, Penicillium, Aspergillus and Alternaria were the most frequent isolates respectively. This study showed that environmental factors affect the number and types of airborne fungi, opportunistic fungi, in Isfahan. Sampling location was an important factor: there were more fungal colony counts in the centre and populated locations of the city than in the less populated and rural areas. Regarding collection time, the most colony counts were obtained from samples collected at mid-day and the least in the morning. This study indicated that the incidence of airborne fungi with clinical significance had a direct relationship with the variation of environmental conditions. The results of the present study contribute towards a better understanding of the pattern of occurrence of airborne fungi, and may be useful for allergists, clinicians and epidemiologists.