Background Compared to soil or aquatic ecosystems, the atmosphere is still an underexplored environment for microbial diversity. Besides its ecological importance, the spatial and temporal characterization of aerosolized microorganisms is relevant for understanding allergy and disease outbreaks, especially in highly populated cities. Results In this study, we surveyed the composition, variability and sources of microbes (bacteria and fungi) in the near surface atmosphere of a highly populated area, spanning ~ 4,000 Km 2 around the city center of Madrid (Spain), in different seasonal periods along two years. We found a core of abundant bacterial genera robust across space and time, most of soil origin, while fungi were more sensitive to environmental conditions. Microbial communities showed clear seasonal patterns driven by variability of environmental factors, mainly temperature and accumulated rain, while local sources played a minor role. We also identified taxa in both kingdoms characteristic of seasonal periods, but not of specific sampling sites or plant coverage. Conclusions The present study suggests that the near surface atmosphere of urban environments constitutes a stable ecosystem, with a relatively homogenous composition, modulated by climatic variations. As such, it contributes to our understanding of the long-term changes associated to the human exposome in the air of highly populated areas.