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Simple Summary: Reproductive disorders in ruminants may be related to bacterial and protozoan agents. Several studies have been carried out to determine the spreading of Brucella spp., Chlamydia abortus, Coxiella burnetii, Salmonella enterica, Listeria monocytogenes, Neospora caninum, and Toxoplasma gondii in domestic ruminants but not in deer. These pathogens are not only a problem for pregnant females because they may cause systemic infections in all animals. Furthermore, considering that all these bacteria as well as T. gondii are zoonotic agents, monitoring wild ruminants is pivotal to verify changes in the epidemiological scenario from a One Health perspective, too. Bacterial and protozoan agents can determine abortion and other reproductive disorders in domestic ruminants, but data regarding their occurrence in wild ruminants are scanty worldwide, including in Italy. The aim of this retrospective study was to verify the occurrence of the main bacterial and protozoan abortive agents in 72 spleen samples previously collected from roe deer (Capreolus capreolus) living in mountain areas of Central Italy. All samples were collected and submitted to DNA extraction for other investigations. Molecular analyses were carried out on the DNA samples to detect Brucella spp., Chlamydia abortus, Coxiella burnetii, Salmonella enterica, Listeria monocytogenes, Neospora caninum, and Toxoplasma gondii. Three (4.16%) roe deer resulted PCR positive for C. burnetii and one (1.38%) for T. gondii. These findings suggest that roe deer living in the investigated areas do not act as important reservoirs of the searched agents. However, the tested animals lived in a closed area without contact with domestic animals that are usually involved in the epidemiology of the investigated pathogens. Monitoring of wild ruminants is pivotal to verify changes in the epidemiological scenario from a One Health perspective, too. [ABSTRACT FROM AUTHOR]