Hibernation in mammals entails profound physiological changes that are known to impact host-associated microbial communities, yet its effects on the gut microbiota of synanthropic bats remain underexplored. In this study, we investigated the gut bacterial composition and diversity of Nyctalus noctula before and during hibernation using high-throughput 16S rRNA amplicon sequencing. Fecal samples from individually banded bats were collected under controlled conditions at a rehabilitation center and analyzed for alpha and beta diversity, as well as differential taxonomic abundance. Hibernation was associated with a marked reduction in microbial diversity according to the Shannon and Simpson indices and a distinct restructuring of gut communities based on the Bray–Curtis dissimilarity index. Active bats exhibited a diverse microbiota enriched in facultative anaerobes, including Lactococcus, Enterococcus, and Escherichia–Shigella, while hibernating individuals were dominated by obligate anaerobes, such as Romboutsia and Paeniclostridium. These findings suggest a contraction and functional specialization of the gut microbiota during torpor, potentially reflecting adaptations to fasting, hypothermia, and reduced gut motility. Our results demonstrate that the bat’s gut microbiome is highly responsive to physiological status and underscore the importance of microbial ecology for understanding the host’s energy balance and health under seasonal contexts.