The microbiological patterns of the passenger cabins of urban public transportation by the example of 41 buses from 16 routes was described for the first time. According to orographic features of the city of Nizhny Novgorod, all the bus routes were divided into three model groups designated as “High-bank,” “Low-bank,” and “Interdistrict.” The abundance of microorganisms and the species composition were estimated using an Autoflex MALDI mass spectrometer (Bruker Daltonics, Germany) and the BioTyper software. In the samples collected, 85 microbial species were detected, identified, and assigned to one of the following three groups according to their degree of pathogenicity: (1) opportunistic pathogens often causing infectious diseases; (2) opportunistic pathogens rarely causing infectious diseases, and (3) nonpathogenic microorganisms. To characterize the ecological structure of the microbial community of urban public transport, two similarity indices for the microbiocoenoses studied, the Sorensen binary index and the multidimensional Koch biotic dispersion index, were calculated. The species composition was analyzed at two hierarchical levels of the bus route groups (the intragroup and intergroup ones). At the intragroup level, a small number (1–3) of common species represented by Staphylococcus epidermidis, S. haemolyticus, and Acinetobacter lwoffii were found in each route group, which corresponds to a low Koch index value (0.1). At the intergroup level, 15 common species of microorganisms were found, which corresponds to the value of the Koch index calculated for the three route groups (0.26), i.e., 26% of species were common for the three route groups analyzed. Species of three groups of microorganisms were most comprehensively represented in the “Interdistrict” group: 45% species of Group 1, 53.8% species of Group 2, and 58.1% species of Group 3. Community structure analysis carried out using ecological indices (Shannon species diversity index, Simpson index of dominance, Margalef species richness index, and Pielou species evenness index) showed that an increase in the species diversity of microbial communities was accompanied by a regular decrease in dominance and an increase in evenness. Analysis of the group of dominant species identified in the buses of the three route groups revealed that the species similarity of the dominant species, estimated by the value of the Koch index, was only 0.14, which could be explained by the presence of only one common species (Acinetobacter lwoffii). Studies of the microbial community of urban transportation present a perspective for solving the sanitary, hygienic, and environmental aspects of the integrated problem of ensuring the ecological safety of the urban environment.