This study investigates the antiviral activity of N-methylisatin-β-thiosemicarbazone derivatives, specifically SCH 16, against Japanese encephalitis virus (JEV), dengue-2 (Den-2), and West Nile virus (WNV). SCH 16 was found to completely inhibit JEV and WNV replication in vitro, with IC50 values of 16 μg/ml and 4 μg/ml, respectively. However, it showed no antiviral activity against Den-2. In vivo, SCH 16 at 500 mg/kg body weight, administered orally twice daily, completely prevented mortality in mice challenged with 50LD50 JEV. The compound inhibited JEV replication at the level of early protein translation. The therapeutic index (TI) of SCH 16 was 5 for JEV and 19 for WNV, indicating moderate to high activity. The study also evaluated the in vivo efficacy of SCH 16 in mice using intracerebral and intraperitoneal challenge models. In the intraperitoneal model, SCH 16 showed dose-dependent protection against JEV, with 50% protection at 400 mg/kg and complete protection at 500 mg/kg. No viable virus or viral antigen was detected in the brains of surviving mice, suggesting effective inhibition of JEV replication. The results indicate that SCH 16 is a promising candidate for further development as an antiviral agent against JEV and WNV. The study highlights the potential of isatin derivatives as antiviral agents and suggests that further research is needed to explore their mechanisms of action and optimize their therapeutic potential.This study investigates the antiviral activity of N-methylisatin-β-thiosemicarbazone derivatives, specifically SCH 16, against Japanese encephalitis virus (JEV), dengue-2 (Den-2), and West Nile virus (WNV). SCH 16 was found to completely inhibit JEV and WNV replication in vitro, with IC50 values of 16 μg/ml and 4 μg/ml, respectively. However, it showed no antiviral activity against Den-2. In vivo, SCH 16 at 500 mg/kg body weight, administered orally twice daily, completely prevented mortality in mice challenged with 50LD50 JEV. The compound inhibited JEV replication at the level of early protein translation. The therapeutic index (TI) of SCH 16 was 5 for JEV and 19 for WNV, indicating moderate to high activity. The study also evaluated the in vivo efficacy of SCH 16 in mice using intracerebral and intraperitoneal challenge models. In the intraperitoneal model, SCH 16 showed dose-dependent protection against JEV, with 50% protection at 400 mg/kg and complete protection at 500 mg/kg. No viable virus or viral antigen was detected in the brains of surviving mice, suggesting effective inhibition of JEV replication. The results indicate that SCH 16 is a promising candidate for further development as an antiviral agent against JEV and WNV. The study highlights the potential of isatin derivatives as antiviral agents and suggests that further research is needed to explore their mechanisms of action and optimize their therapeutic potential.