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Humans distribute a wide range of microorganisms around building interiors, and some of these are potentially pathogenic. Recent research established that humans are the main drivers of the indoor microbiome and up to now significant literature has been produced about this topic. Here we analyzed differences in bacterial composition between men’s and women’s restrooms and other common areas within the same public building. Bacterial DNA samples were collected from restrooms and halls of a three-floor building from the Federal University of Pampa, RS, Brazil. The bacterial community was characterized by amplification of the V4 region of the 16S rRNA gene and sequencing. Throughout all samples, the most abundant phylum was Proteobacteria, followed by Actinobacteria, Bacteroidetes and Firmicutes. Beta diversity metrics showed that the structure of the bacterial communities were different among the areas and floors tested, however, only 6-9% of the variation in bacterial communities was explained by the area and floors sampled. A few microorganisms showed significantly differential abundance between men’s and women’s restrooms, but in general, the bacterial communities from both places were very similar. Finally, significant differences among the microbial community profile from different floors were reported, suggesting that the type of use and occupant demographic within the building may directly influence bacterial dispersion and establishment. [ABSTRACT FROM AUTHOR]