N-methylisatin-β-thiosemicarbazone (MIBT) derivative SCH 16 has been shown to inhibit the replication of Japanese Encephalitis Virus (JEV) and West Nile Virus (WNV) in vitro and in vivo. Among 14 MIBT derivatives tested, only SCH 16 exhibited significant antiviral activity against JEV and WNV, with an IC50 of 16 μg/ml and 4 μg/ml, respectively. In a mouse model, SCH 16 administered orally at 500 mg/kg body weight completely prevented mortality in mice challenged with 50LD50 JEV. The mechanism of action suggests that SCH 16 inhibits JEV replication at the early stage of protein translation. These findings indicate that SCH 16 has potential as a therapeutic agent for JEV infection.N-methylisatin-β-thiosemicarbazone (MIBT) derivative SCH 16 has been shown to inhibit the replication of Japanese Encephalitis Virus (JEV) and West Nile Virus (WNV) in vitro and in vivo. Among 14 MIBT derivatives tested, only SCH 16 exhibited significant antiviral activity against JEV and WNV, with an IC50 of 16 μg/ml and 4 μg/ml, respectively. In a mouse model, SCH 16 administered orally at 500 mg/kg body weight completely prevented mortality in mice challenged with 50LD50 JEV. The mechanism of action suggests that SCH 16 inhibits JEV replication at the early stage of protein translation. These findings indicate that SCH 16 has potential as a therapeutic agent for JEV infection.