Simple SummaryPublic transport facilities, including subway systems, provide the most suitable conditions for the transfer of microorganisms between people and the environment, contributing to the pathogenic potential of the urban habitat. Investigation of microbiome diversity and description of its characteristic properties, e.g., antibiotic-resistance profiles, leads to understanding of these interactions. In this study, we aimed to conduct an extended analysis of the bioaerosol and surface microbiome of the Moscow subway, using 16S rRNA gene sample sequencing and classical microbiology methods. The microbiomes of two subway stations (Novokosino and Cherkizovskaya) were reconstructed which differ in terms of passenger traffic and duration of exploitation. It was shown that most bacterial genera were ubiquitous; however, the unique genera were presented in aerosol samples. The relatively older Cherkizovskaya station possessed greater diversity in antibiotic resistance among the identified microorganisms compared to Novokosino station. We also provided a comparative analysis of these results with the previously published data, which allowed us to identify the distribution of microorganisms associated with the human microbiome and the environment regardless of the seasonal fluctuations. The obtained results provide valuable information on the diversity of bacterial communities in the Moscow subway, one of the most socially important facilities in metropolitan areas.The subway is one of the most actively used means of transport in the traffic infrastructure of large metropolitan areas. More than seven million passengers use the Moscow subway every day, which promotes the exchange of microorganisms between people and the surrounding subway environment. In this research, a study of the bacterial communities of two Moscow subway stations was conducted and the common subway microbiome was determined. However, there were differences in microbiological and antibiotic-resistance profiles, depending on the station. The station’s operational period since opening correlated with the taxonomic diversity and resistance of the identified bacteria. Moreover, differences between aerosol and surface bacterial communities were found at the two subway stations, indicating the importance of diversified sampling during the microbiome profiling of public areas. In this study, we also compared our data with previously published results obtained for the Moscow subway. Despite sample collection at different stations and seasons, we showed the presence of 15 common genera forming the core microbiome of the Moscow subway, which represents human commensal species, as well as widespread microorganisms from the surrounding environment.