Neospora caninum is an apicomplexan (family: Sarcocystidae) protozoan parasite with a global distribution. In the N. caninum life cycle, dogs and other related canids are the definitive hosts, while other animals such as water buffaloes (Bubalus bubalis) constitute the intermediate host for this parasite. In many countries, the water buffalo is of high economic importance, providing valuable high-quality products for human needs. Although knowledge concerning the prevalence of this parasite in intermediate animal host populations is crucial, data from water buffalo are scarce. Keeping this in mind, the aim of this study was to examine the presence and assess the prevalence rates of N. caninum infection in water buffaloes in Northern Greece, where this animal husbandry sector started to raise, as well as to determine associated risk factors, with the application of a commercially available Neospora ISCOM ELISA test kit, developed for the detection of antibodies against N. caninum in milk samples The study was conducted during January–June 2023 in a total of 172 individual raw milk samples, collected from dairy buffaloes, reared under a semi extensive system, in three different farms. Information on the status of N. caninum infection in buffaloes from Greece was so far unknown with a lack of epidemiological or risk factors associated. For the detection of N. caninum, the commercially available Neospora ISCOM ELISA test kit (SANOVIR ®, Svanova Biotech AB, Uppsala, Sweden) was utilized. Results demonstrated the presence of N. caninum in water buffaloes from Greece for the first time. All positive N. caninum animal were asymptomatic, with absence of any disease signs. The overall prevalence of infection was 22.10%, whereas the main risk factors include the presence of dogs as well as the low biosecurity measures.