The aim of the study was to determine the seroprevalence of anti-Borrelia burgdorferi antibodies in a population of Turkish dogs and horses, as well as to compare the sensitivity of novel flow-cytometry-based borreliacidal antibody test (BAT) with ELISA assay. Serum samples collected from 400 dogs and 300 horses were tested with enzyme-linked protein A/G assay (ELPAGA), using Borrelia whole cell antigens. ELPAGA test showed 93 dogs (23.2%) and 18 horses (6%) serologically positive for anti-Borrelia antibodies. In parallel testing of sera with BAT, we found 27.75% positive dogs and 6.33% positive horses. When the results of these serological testes were compared with the health status of the animals, the most common clinical signs noticed in dogs were skin manifestations, urinary tract disorder and anemia; however, no clinical symptoms were observed in horses positive for the anti-Borrelia antibodies. This is a first time that seroprevalence of Lyme disease in dogs and horses has been reported from Turkey, as well as the use of novel BAT in animals.