| Summary: | Wild small mammals are the most common reservoirs of pathogenic microorganisms that can cause zoonotic diseases. The aim of the study was to detect antibodies related to <i>Borrelia burgdorferi</i> sensu lato, <i>Coxiella burnetii,</i> and <i>Francisella tularensis</i> in wild small mammals from the Czech Republic. In total, sera or heart rinses of 211 wild small mammals (168 <i>Apodemus flavicollis</i>, 28 <i>Myodes glareolus</i>, 9 <i>A. sylvaticus</i>, and 6 <i>Sorex araneus</i>) were examined by modified enzyme-linked immunosorbent assay. Antibodies related to <i>B.</i><i>burgdorferi</i> s.l., <i>C. burnetii</i>, and <i>F. tularensis</i> were detected in 15%, 19%, and 20% of animals, respectively. The prevalence of <i>B. burgdorferi</i> and <i>F. tularensis</i> statistically differed in localities and <i>F. tularensis</i> also differed in sex. Antibodies against 2–3 pathogens were found in 17% of animals with a higher prevalence in <i>M. glareolus</i>. This study brings new data about the prevalence of the above-mentioned pathogens.
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