For the first time, a mycological analysis of outdoor urban environment (air, leaves, sealed surfaces) was carried in the cities of subarctic (Murmansk) and temperate (Moscow) climatic zones. The chemical composition of dust deposited on leaves of dominant tree species was taken as an indicator of the air quality. Assessment of the complex impact of factors (climate zone, type of substrate, anthropogenic load) on the quantitative and qualitative parameters of mycobiome was performed. Compared to Moscow, Murmansk was characterized by an increased number and concentrations of pollutants in the deposited dust. The number of culturable airborne fungi in Murmansk was substantially lower than in Moscow. Half of the species belonged to the opportunistic in both cities. Most dangerous opportunistic fungi were absent in the air of recreational zones but present on leaves surface and in road dust in all assessed zones of the cities. Dust chemical composition affected the diversity of fungi species. While the relationship of biological parameters with concentration of potentially toxic metals was generally negative, Cd increased the fraction of opportunistic fungi in road dust. The study revealed an importance of substrate in determining the sensitivity of outdoor mycobiome to pollution and highlighted its biological characteristics sensitive to climate.