Hibernation is an extraordinary phenomenon evolved in many animals including some mammals, allowing them to survive unfavorable environmental conditions. This period represents a phase of fasting, which is known to affect the gut microflora in nonhibernating mammals. Since during hibernation the physiological parameters (e.g., body temperature) differ from values in nonhibernating individuals, the food starvation is not the only parameter affecting the gut microflora. However, little is known about gut microflora in hibernating mammals. This study is focused on the examination of the gut microflora of Mediterranean horseshoe bat (Rhinolophus euryale) during hibernation. Faecal samples were collected during the winter from November 2014 to March 2015 and subsequently subjected to cultivation, non-cultivation analyses and morphological examination. Cultivation analysis revealed that the numbers of total cultivable bacteria, enterobacteria and enterococci in the faecal samples decreased during the hibernation and restored to pre-hibernation numbers at the end of hibernation. Results of non-cultivation analysis showed time-dependent (but surprisingly almost not individual-specific) changes in the gut microflora and decrease in bacterial variability dependent on hibernation stage. Changes in microflora were accompanied by changes in faecal content identified by morphological analysis. Our results demonstrate that hibernation affects the gut microflora of bats in significant degree in both quantitative and qualitative scale.