Objective: To determine the prevalence of vector-borne bacteria and protozoa in hunting dogs living in Central Italy. Methods: Molecular testing was executed on DNA which was extracted from blood specimens collected from 117 asymptomatic dogs to detect Anaplasma phagocytophilum, Babesia canis (B. canis), Bartonella spp., Coxiella burnetii (C. burnetii), Ehrlichia canis, Hepatozoon canis, and Leishmania infantum. Results: A total of 48 dogs (41.0%) were infested by Ixodes ricinus and Rhipicephalus sanguineus ticks. Tick-borne infections were observed in 64 (54.7%) animals. More in detail, 38 dogs (32.5%) screened positive for Hepatozoon canis, 24 (20.5%) for Bartonella vinsonii subsp. berkhoffii, 20 (17.1%) for Leishmania infantum, 6 (5.1%) for C. burnetii, 5 (4.3%) for B. canis (3 B. canis vogeli and 2 B. canis canis), 3 (2.5%) for Anaplasma phagocytophilum, and 2 (1.7%) for Ehrlichia canis. Mixed infection by 2 agents occurred in 17 (14.5%) subjects, by 3 agents in 7 (6.0%) dogs, and by 4 agents in 1 (0.9%) animal. Conclusions: The results demonstrated that several vector-borne pathogens were circulating in this region and dogs infected by these agents were usually asymptomatic. A relevant finding was the presence of DNA of C. burnetii, a severe zoonotic agent, in the 5.1% of tested dogs, which can be source of infection for their owners not only through tick bites, but also directly with urine, feces and birth products.