In autumn 2024, a highly pathogenic avian influenza (HPAI) H5N1 virus outbreak occurred in a mixed-species backyard poultry flock in central Serbia, comprising hens, ducks, and geese. After the sudden death of two hens, a thorough diagnostic investigation was initiated. Gross examination of the affected hens revealed lesions typical of HPAIV infection, including severe cyanosis of the comb and head skin, and diffuse hemorrhagic lesions of internal organs. On-site investigation included assessment of the remaining birds, environmental sampling, and a biosecurity survey specific to backyard flocks. Although asymptomatic, ducks and geese (n = 23) tested positive for avian influenza by both oropharyngeal and cloacal swabs, as well as by serological testing. The biosecurity assessment highlighted major deficiencies, including uncontrolled access to a nearby canal frequented by wild waterfowl. Overall, virus introduction at the domestic-wild bird interface was considered the most epidemiologically plausible hypothesis. However, this pathway could not be confirmed because wild birds were not sampled and viral genome sequencing was not performed.