In central Europe, at least three flaviviruses circulate among vectors and vertebrate hosts. West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses maintained in the nature by enzootic cycle between mosquitoes and birds. Tick-borne encephalitis virus (TBEV) is a flavivirus causing annual human cases in Slovakia. The aim of this study is the prevalence assessment of flavivirus infections in horses (n = 145) and birds (n = 109) by enzyme-linked immunosorbent assay (ELISA) and confirmation by neutralization test (VNT). WNV antibodies have been detected in 11.7% of tested horses and 11.9% of tested birds and confirmed in 6.9% of horse and 9.2% of bird samples. None of the WNV seropositive or dubious horses had WNV IgM (ELISA), and none of the tested horses had USUV neutralizing antibodies. Autochthonous WNV infections have been confirmed in 16.7% of horses without international travelling history. Most of them were from western Slovakia with known endemic WNV transmission. An autochthonous WNV infection in a horse from highland area of Kremnické vrchy (central Slovakia) with unknown data of WNV circulation and without travelling history was detected. TBEV antibody was detected in 6.2% of horses and in 3.4% has been confirmed. In two horses, WNV and TBEV infection could not be distinguished. Confirmed WNV seropositive were eight raptors showing nonspecific signs or suffering from trauma, one white stork, and one house sparrow. The sparrow was caught in a locality in eastern Slovakia, where WNV RNA had been previously detected in sparrows. USUV neutralizing antibodies were present in pooled sample from four Eurasian great tits. Because of insufficient volume, TBEV VNT was not carried out in birds. Results further prove the endemicity of WNV and other vector-borne flaviviruses in natural and accidental hosts in Slovakia, giving better insight in flavivirus epidemiology in European countries in general.