Airborne fungi are widely distributed in the environment and originate from various sources like soil, plants, decaying organic matter, and even indoor environments. Exposure to airborne fungal spores can cause allergic reactions, asthma, and respiratory infections. Certain fungi can cause serious infections, particularly in individuals with weakened immune systems. An aerobiological study was conducted to detect airborne fungi using metagenomics in three areas of Mexico City, each representing different degrees of urbanization, during the dry and rainy seasons of 2017. Seasonality showed a significant role in the composition of airborne fungi. Ascomycota predominated in the three areas sampled during dry and rainy seasons, with the genera and presenting the highest relative abundances across seasons. The Agaromycetes showed an increase during the rainy season. Regarding the areas, the north and center sites showed similar meteorological conditions and fungal community composition patterns. According to the Shannon and Simpson indices, the south area showed a greater species diversity during the dry season. These insights into the interactions between urbanization, seasonality, and airborne microbial communities could contribute to more effective urban management, reduced health risks, and the promotion of sustainable development.