This study delves into the synthesis, characterization, and biological evaluation of novel nickel(II) complex with isatin-based thiosemicarbazone (TSC), aimed at exploring their potential in cancer therapy. Employing a blend of experimental methodologies and computational tools, including density functional theory (DFT) calculations, molecular docking, and dynamics simulation, we investigated the stability, electronic properties, and biological effects of these compounds. Analysis via spectroscopy validated successful synthesis, offering insights into their structural characteristics, while X-ray diffraction analysis affirmed their crystalline structure. DFT calculations were utilized to assess electronic structure optimization, HOMO-LUMO energy gaps, and global parameters, with molecular modeling revealing bond angles, bond lengths, and pertinent quantum chemical factors using the DFT method. Additionally, molecular docking and MD simulations were conducted to forecast binding modes and interactions between the ligands and the SARS-CoV-2 main protease (MPro). Computational findings underscored advantageous electronic structures and potential reactivity, supported by molecular docking studies showing robust binding affinities towards vital biological targets such as the SARS-CoV-2 main protease, human ACE-2 enzyme, and VEGFR2. Furthermore, the anticancer potential of these compounds was assessed via in vitro assays on diverse cancer cell lines, unveiling selective cytotoxicity, and encouraging therapeutic prospects. © 2025 Elsevier B.V., All rights reserved.