Background: Long-term or frequent use of currently approved anti-influenza agents has resulted in the emergence of drug-resistant viruses, necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few subtypes (e.g., seasonal H1N1 and H3N2) of influenza A virus (IAV). However, there were no reports of its inhibitory effects on the other types of influenza virus. Therefore, in this study, we investigated the inhibitory effects of aprotinin against a wide range of influenza viruses in vitro and in vivo. Methods: We tested the antiviral activity of aprotinin in Madine-Darby canine kidney (MDCK) cells against seasonal human IAVs, avian influenza viruses with zoonotic potential, oseltamivir-resistant IAVs, and influenza B virus. We also tested the antiviral activity of aprotinin against A/PR/8/34 (H1N1) virus in a mouse model. Results: Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Conclusions: Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.