Due to the notable biological activity of isatin hydrazone compounds, we synthesized and structurally characterized three arylidene isatin hydrazone derivatives (3a-3c) utilizing the SBA-Pr-N-Is-Bu-SO₃H nanocatalyst. A comparative investigation was performed through an integrated approach combining experimental assays and theoretical modeling. In vitro biological assays performed on normal umbilical vein endothelial cells (HUVECs) and breast cancer cell lines (MCF-7 and MDA-MB-231) revealed noteworthy anticancer activity, with all derivatives significantly reducing cancer cell viability in a concentration- and time-dependent manner. Furthermore, compound 3c exhibited the most potent antioxidant effect, achieving up to 80% inhibition at higher concentrations. Biomolecule (DNA and BSA) interaction studies were performed utilizing UV-Vis spectroscopic analysis and molecular docking simulations. The values of K_app for the interaction of compounds 3a (1.01 × 10⁴ M^− 1), 3b (1.17 × 10⁴ M^− 1), and 3c (2.03 × 10⁴ M^− 1), along with docking simulations, indicate that the studied compounds are likely to bind to CT-DNA via a groove-binding mechanism. Lipophilicity assessments demonstrated that compounds 3b and 3c had significantly higher log P values than cisplatin, indicating enhanced lipid affinity and superior cellular membrane permeability potential. Comprehensive computational analyses, including DFT/TD-DFT, topology analysis, and ADME-Tox profiling, were employed to further validate the observed biological activity and pharmacokinetic potential. The convergence of computational insights and experimental findings provides robust validation for the potential of these compounds as promising anticancer agents.