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Wild small mammals and ticks play an important role in maintaining and spreading zoonoses in nature, as well as in captive animals. The aim of this study was to monitor selected agents with zoonotic potential in their reservoirs and vectors in a zoo, and to draw attention to the risk of possible contact with these pathogens. In total, 117 wild small mammals (rodents) and 166 ticks were collected in the area of Brno Zoo. Antibodies to the bacteria <i>Coxiella burnetii</i>, <i>Francisella tularensis</i>, and <i>Borrelia burgdorferi</i> s.l. were detected by a modified enzyme-linked immunosorbent assay in 19% (19/99), 4% (4/99), and 15% (15/99) of rodents, respectively. Antibodies to <i>Leptospira</i> spp. bacteria were detected by the microscopic agglutination test in 6% (4/63) of rodents. Coinfection (antibodies to more than two agents) were proved in 14.5% (15/97) of animals. The prevalence of <i>C. burnetii</i> statistically differed according to the years of trapping (<i>p</i> = 0.0241). The DNAs of <i>B. burgdorferi</i> s.l., <i>Rickettsia</i> sp., and <i>Anaplasma phagocytophilum</i> were detected by PCR in 16%, 6%, and 1% of ticks, respectively, without coinfection and without effect of life stage and sex of ticks on positivity. Sequencing showed homology with <i>R. helvetica</i> and <i>A. phagocytophilum</i> in four and one positive samples, respectively. The results of our study show that wild small mammals and ticks in a zoo could serve as reservoirs and vectors of infectious agents with zoonotic potential and thus present a risk of infection to zoo animals and also to keepers and visitors to a zoo.