Background: Despite abundance of small mammals in Serbia, there is no information on their role in the epidemiology of tick-borne diseases (TBDs). This retrospective study aimed to identify different tick-borne pathogens (TBPs) in small mammals in Serbia collected during 2011. Materials and Methods: A total of 179 small mammals were collected from seven different localities in Serbia. The five localities belong to the capital city of Serbia-Belgrade: recreational areas-Ada Ciganlija, Titov gaj, and Košutnjak as well as mountainous suburban areas used for hiking-Avala and Kosmaj. The locality Veliko Gradište is a tourist place in northeastern Serbia, whereas the locality Milošev Do is a remote area in western Serbia with minor human impact on the environment. Results: The results of the presented retrospective study are the first findings of Rickettsia helvetica , Rickettsia monacensis , Neoehrlichia mikurensis , Borrelia afzelii , Borrelia miyamotoi , Babesia microti , Hepatozoon canis, and Coxiella burnetii in small mammals in Serbia. The presence of R. helvetica was confirmed in two Apodemus flavicollis , the presence of one of the following pathogens, R. monacensis , B. afzelii , H. canis , Ba. microti , and N. mikurensis was confirmed in one A. flavicollis each, whereas the presence of B. miyamotoi was confirmed in one Apodemus agrarius . Coinfection with B. afzelii and Ba. microti was confirmed in one A. flavicollis . DNA of C. burnetii was detected in 3 of 18 pools. Conclusions: The results confirm that detected pathogens circulate in the sylvatic cycle in Serbia and point to small mammals as potential reservoir hosts for the detected TBPs. Further large-scale studies on contemporary samples are needed to clarify the exact role of particular small mammal species in the epidemiology of TBDs caused by the detected pathogens.