In order to investigate the prevalence of tick-borne diseases, equine piroplasmosis, equine granulocytic anaplasmosis and Lyme borreliosis in Central Italy, blood samples from 300 horses were analyzed for the presence of antibodies against Babesia caballi, Theileria equi, Anaplasma phagocytophilum and Borrelia burgdorferi using the IFAT. The blood samples were also subjected to PCR assays in order to detect pathogen DNA. A total of 78 (26.0%) and 123 (41.0%) horses were found to be seropositive for B. caballi and T. equi, respectively, while 41 (13. 4%) and 21 (7.0%) horses were, respectively, seropositive for A. phagocytophilum and B. burgdorferi. Seropositivity for more than one agent was detected in 76 horses using IFAT. The most common association observed was between T. equi and B. caballi (14.7%). In addition, 54 horses (18.0%) were found to be positive for one or more tick-borne pathogens (TBPs) using PCR testing. Among these, 28 (9.3%) harbored single infections, while 26 (8.7%) were found to be co-infected with two or more pathogens. The correlation (K value) between IFAT and PCR results was 0.32 for T. equi, 0.34 for B. caballi, 0.62 for B. burgdorferi and 0.48 for A. phagocytophilum, reflecting an unprecedented degree of multiple exposures to TBPs in horses.