The indigenous intestinal microbiota forms a dynamic ecosystem, the equilibrium of which is essential for the host's well-being. Disturbance of the small intestinal microbiota have been implicated as a risk factor of small intestinal disease. Tylosin, a bacteriostatic macrolide antibiotic, is commonly recommended for the treatment of intestinal disorders such as small intestinal bacterial overgrowth or tylosin-responsive diarrhea in dogs. However, there are only few data available about the influence of tylosin on the composition of the small intestinal microbiota. The aim of this study was to evaluate the dynamics of the jejunal microbiota after administration of tylosin at a dose corresponding to routine therapeutic. Five healthy laboratory beagles with permanent jejunal fistula located approximately 60cm distal to the pylorus were included into the study. Tylosin was administered at a dose of 20-22 mg/kg/day for a period of 14 consecutive days. Samples of jejunal juice were collected through the fistula on day 0 (before tylosin administration), day 14, and day 28 (14 days after withdrawal of tylosin). For evaluation of changes in the levels of total anaerobic and lactic acid bacteria (LAB) in jejunal chyme, samples were cultivated anaerobically on Brucella and MRS media, respectively. From two highest dilutions 20 colonies of LAB were randomly selected, and identified to species level using library based numerical analysis of 16 and 23 S rRNA genes RFLP. Inter-individual variations in the levels of both total anaerobic bacteria and LAB were detected. LAB levels were not markedly altered during tylosin treatment. However, the administration of tylosin increased the proportion of enterococci, particularly Enterococcus fecalis. During tylosin treatment the proportion of enterococci within all LAB was 98% compared to 9.5% before and after tylosin administration. The results support the concept that tylosin may promote the growth of beneficial commensal bacteria such as enterococci, i.e. strains that are known to have probiotic characteristics.