The main protease (M or 3CL or nsp5) of SARS-CoV-2 is crucial to the life cycle and pathogenesis of SARS-CoV-2, making it an attractive drug target to develop antivirals. This study employed the virtual screening of a few phytochemicals, and the resultant best compound, Scopoletin, was further investigated by a FRET-based enzymatic assay, revealing an experimental IC of 15.75 µM. The impact of Scopoletin on M was further investigated by biophysical and MD simulation studies. Fluorescence spectroscopy identified a strong binding constant of 3.17 × 10 M⁻ for Scopoletin binding to M, as demonstrated by its effective fluorescence quenching of M. Additionally, CD spectroscopy showed a significant reduction in the helical content of M upon interaction with Scopoletin. The findings of thermodynamic measurements using isothermal titration calorimetry (ITC) supported the spectroscopic data, indicating a tight binding of Scopoletin to M with a K of 2.36 × 10 M. Similarly, interaction studies have also revealed that Scopoletin forms hydrogen bonds with the amino acids nearest to the active site, and this has been further supported by molecular dynamics simulation studies. These findings indicate that Scopoletin may be developed as a potential antiviral treatment for SARS-CoV-2 by targeting M.