The role of bats in the global microbial ecology no doubt is significant due to their unique immune responses, ability to fly, and long lifespan, all contributing to pathogen spread. Some of these animals hibernate during winter, which results in the altering of their physiology. However, gut microbiota shifts during hibernation is little studied. In this research, we studied cultivable gut microbiota composition and diversity of Nyctalus noctula before, during, and after hibernation in a bat rehabilitation center. Gut microorganisms were isolated on a broad spectrum of culture media, counted, and identified with mass spectrometry. Linear modeling was used to investigate associations between microorganism abundance and N. noctula physiological status, and alpha- and beta-diversity indexes were used to explore diversity changes. As a result, most notable changes were observed in Serratia liquefaciens, Hafnia alvei, Staphylococcus sciuri, and Staphylococcus xylosus, which were significantly more highly abundant in hibernating bats, while Citrobacter freundii, Klebsiella oxytoca, Providencia rettgeri, Citrobacter braakii, and Pedicoccus pentosaceus were more abundant in active bats before hibernation. The alpha-diversity was the lowest in hibernating bats, while the beta-diversity differed significantly among all studied periods. Overall, this study shows that hibernation contributes to changes in bat cultivable gut microbiota composition and diversity.