The aim of our study was to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum in small mammals and ticks using polymerase chain reaction and to gain information about the prevalence and possible coexistence of these pathogens at a selected site in Hungary. Two hundred seventy-seven small mammals were trapped in South-Eastern Hungary during 2009. Tissue samples and a total of 831 ectoparasites (Ixodes ricinus, Ixodes acuminatus, Haemaphysalis concinna, Ctenophtalmus assimilis, and Nosopsyllus fasciatus) were collected from small mammals. One thousand one hundred and six I. ricinus and 476 H. concinna were collected from the vegetation during the investigation. Neither A. phagocytophilum nor B. burgdorferi s.l. was detected in any of the mammal tissue samples. A. phagocytophilum was not found in ticks collected from small mammals. Very low minimum prevalence was found for all pathogens (0.62% for Borrelia afzelii in ticks collected from small mammals, and 0.57%, 0.06%, and 0.19% for A. phagoctyophilum, B. afzelii, and Borrelia garinii, respectively, in questing ticks). The present study is the first report of borreliae from I. acuminatus and H. concinna from Hungary.