BACKGROUND: The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains unknown. However, it is likely that the virus spillover occurred from an animal reservoir to humans. Identifying animal species susceptible to SARS-CoV-2 is crucial for understanding cross-species transmission to humans. This study distinguishes itself by focusing on the susceptibility of the European bison (Bison bonasus), an endangered species, to SARS-CoV-2. The objective of this study was to investigate the occurrence of SARS-CoV-2 antibodies in a substantial number (n = 238) of both free-living and captive Polish European bison using an in-house ELISA method and virus neutralization test (VNT). RESULTS: The seroprevalence of SARS-CoV-2 infection was found to be 1.29% (3/232). None of the seropositive European bison tested positive in the virus neutralization test. All seropositive animals were part of captive herds. CONCLUSIONS: This study represents the first report of SARS-CoV-2 seroprevalence in both free-ranging and captive European bison in Poland. Based on these findings, the European bison appears to be a less susceptible species to SARS-CoV-2. The most probable route of transmission was from humans to European bison, as all seropositive animals belonged to captive herds with contact with indirect human sources, such as tourists and keepers.