Neospora caninum and Toxoplasma gondii are cosmopolite protozoan parasites impacting on human and animal health. In particular, T. gondii commonly infects human beings and all warm-blooded animals, while N. caninum is responsible for bovine abortion and neuromuscular disease in dogs. The aim of the presented survey was to evaluate the occurrence and prevalence of these parasites in the most numerous Italian red deer population. The sera of 60 red deer (Cervus elaphus) inhabiting Central Italy (43°56’N 10°55’E) and killed by selective hunting were examined using an indirect fluorescent antibody test (IFAT) for both N. caninum and T. gondii antibodies. White blood cells (buffy coat) were also checked by PCR and T. gondii DNA was genotyped. Thirteen out of 60 sera (22%) scored positive for Toxoplasma, 17 samples (28%) were Neospora positive. Coinfection was recorded in 5 cases (8%). T. gondii (genotype II) and N. caninum DNA was detected in one and 3 samples of buffy coat, respectively. The presented study is the first to examine the occurrence of these parasites in the most numerous red deer Italian population, confirming this animal species as carrier of the investigated pathogens. These animals spread near human settlements, co-inhabiting with final hosts of T. gondii and N. caninum and could contribute to their transmission to domestic ruminants and humans. In particular, the seroprevalence value for N. caninum was the highest among European records.