The aim of this study was to help improve the management of assisted reproduction of donkeys and to extend the existing information on the fertility of donkeys by qualitative and quantitative assessment of bacterial contamination of the jennies’ genital tract in connection with the perineum formation and oestrous cycle phases. Ten female donkeys were included in the study and were repeatedly rectally palpated and sonographically examined during the oestrous cycle. Samples were taken from the fossa clitoridis and around the cervix for microbiological examination. Each jenny was sampled twice, always at different stages of the cycle, both in the oestrus and in the dioestrus. In addition, samples from the endometrium were taken in oestrus for both microbiological and cytological examination. After collection, the samples were examined in a microbiological laboratory. There were 62 different species of microorganisms found belonging to 19 different bacterial genera. The presence of agents of mares’ infectious metritis Taylorella equigenitalis was not found in any of the samples. The outcome of our study is an initial mapping of the microbial colonization of the endometrium and the genital tract in jennies with correct formation of the perineal region. The hypothesis that the level of bacterial contamination was related to the oestrous cycle was not confirmed. The practical significance of the study lies mainly in the description of the composition, amount and changes of microbiota in healthy individuals during their oestrous cycle, which allows the evaluation of the risk of developing infection during the active oestrous cycle in connection with mating.