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Microbes are abundant inhabitants of the near-surface atmosphere in urban areas. The distribution of microbial communities may benefit or hinder human wellbeing and ecosystem function. Surveys of airborne microbial diversity are uncommon in both natural and built environments and those that investigate diversity are stationary in the city, thus missing continuous exposure to microbes that covary with three-dimensional urban structure. Individuals in cities are generally mobile and would be exposed to diverse urban structures outdoors and within indoor-transit systems in a day. We used mobile monitoring of microbial diversity and geographic information system spatial analysis, across Philadelphia, Pennsylvania, USA in outdoor and indoor-transit (subways and train cars) environments. This study identifies to the role of the three-dimensional urban landscape in structuring atmospheric microbiomes and employs mobile monitoring over ~1,920 kilometers to measure continuous biodiversity. We found more diverse communities outdoors that significantly differ from indoor-transit air in microbial community structure, function, likely source environment, and potentially pathogenic fraction of the community. Variation in the structure of the urban landscape was associated with diversity and function of the near-surface atmospheric microbiome in outdoor samples.