This study investigates the seroprevalence of Greek adult cattle against West Nile virus, Anaplasma ovis and Lehismania infantum. In total, 156 serum samples were examined, drawn from cattle between 2-4 years old. All the examined cattle originated from slaughterhouses of 4 prefectures in Northern Greece (Thessaloniki, Pella, Chalkidiki, Kilkis), in 2 of which (Thessaloniki, Pella) human cases of West Nile virus had been recorded some months before. Thirty out of the 156 (18.6%) samples have tested positive for West Nile virus and fifty-five (35.9%) samples for Anaplasma ovis. All the examined samples tested negative for Leishmania infantum. The prefectures with positive samples against West Nile virus also showed human cases of West Nile virus infections. This should raise questions whether cattle could become markers for West Nile virus activity in high risk areas.