Avian influenza viruses (AIVs) are widely distributed and have a wide range of hosts. Recently, the number of cases of infection associated with the circulation of highly pathogenic avian influenza H5N1 2.3.4.4b has raised concerns about its high transmission capacity in birds and mammals. This study analyzed swabs from bird and mammal species from the coast of Paraná and the northwest region of S?o Paulo, Brazil, for the presence of AIV in animals that did not present clinical or histopathological lesions of infection that indicated the need for molecular characterization during monitoring. Of the 661 animals analyzed, three tested positive, two of which were birds (Sula leucogaster and Thalasseus acuflavidus) while one was a mammal (Otaria flavescens) (0.45%, CI 95%: 0.16–1.33). A complete genome sequence of H5N1 AIV was obtained from a brown booby (Sula leucogaster) from the Paraná coast (GISAID accession number: EPI_ISL_1897537). Our study reinforces the importance of continuous genomic surveillance, especially in AIV hosts that do not show signs of infection, to enhance the One-Health assessment approach.