Influenza A virus is a zoonotic pathogen capable of infecting a wide range of hosts, yet data on infections in wild ruminants are limited. The European bison (Bison bonasus), Europe’s largest terrestrial mammal, had not previously been identified as a host for Influenza A virus. This study aimed to provide the first molecular and serological evidence of H1N1 pandemic influenza A virus (A/H1N1pdm) infection in European bison, contributing to the understanding of Influenza A virus ecology at the wildlife–livestock interface. We retrospectively analysed 335 serum samples collected from European bison between 2017 and 2023 using a commercial ELISA detecting antibodies against all Influenza A virus subtypes. Tissue samples (heart and liver) from ELISA-positive animal were further tested by RT-qPCR targeting hemagglutinin (HA) and neuraminidase (NA) genes of A/H1N1pdm, A/H3N2, and A/H5N1. PCR-positive products were sequenced for confirmation. Seroprevalence of Influenza A virus was 0.3% (1/335; 95% CI: 0.02–1.71). Influenza A virus RNA (A/H1N1pdm) was detected in the heart and liver tissue of a 4-year-old captive male from Borecka Forest, culled in 2018. The animal exhibited pulmonary congestion, emphysema, and posthitis at necropsy. This study provides the first molecular and serological confirmation of H1N1pdm infection in European bison. The findings highlight the potential susceptibility of this species to Influenza A virus and raise questions about possible transmission routes, including spillover from domestic animals or humans.