Coronavirus induced disease-19 caused by SARS-CoV-2 has presented an unprecedented health and economic crisis worldwide. Substantial progress has been made in the last two years to develop vaccines against SARS-CoV-2. Along with vaccines, antiviral drugs constitute the secondary line of defense against the viral pathogen. Viral proteins are considered to be promising targets for designing antiviral drugs. The main protease (M^pro) is known to play a significant role in SARS-CoV-2 replication within the host. Accordingly, in the present study, a series of selenium-containing amino acids, selenopyridines and their respective derivatives, were screened for interaction with M^pro (PDB code: 6LU7) by molecular docking approach. The most potent docked compounds, namely nicotinamide diselenide (Nict₂Se₂) and pyridine diselenide (2-Py₂Se₂) with binding affinities in the range of ∼10⁵ M⁻¹, were subjected to biochemical evaluation. The IC₅₀ values of Nict₂Se₂ and 2-Py₂Se₂ for M^pro inhibition estimated by bioassay were ∼ 516.0 ± 0.02 nM and 69.4 ± 0.03 nM, respectively. The toxicity evaluation in a normal lung fibroblast (WI38) cell line suggested that among the above two compounds, Nict₂Se₂ was much safer for biological applications. The circular dichroism studies and competitive kinetic assay using 5,5-dithio-bis-(2-nitrobenzoic acid) as a substrate suggested that Nict₂Se₂ treatment induced structural deformation of M^pro probably through interacting with a cysteine residue present in its active site. Together, the present investigation proposes that organoselenium compounds comprising aromatic amide moieties connected by a diselenide bridge could be potential candidate molecules for the future design of antiviral drugs specifically against SARS-CoV-2.