The present study aims to explore the anticancer efficacy of Diosmin by inducing mitochondrial-mediated apoptosis in human epidermoid carcinoma cells (Hep-2). This is done by cell line assays and studying crucial inflammatory and apoptotic signaling molecules. The cytotoxicity property of Diosmin was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Marker expression study was done by western blotting for studying apoptotic markers like Bax, Bcl-2, p53, Bak, and Bcl-xl, proinflammatory cytokine (TNF-α), interleukins (IL-1, IL-6, IL-8), and signal transduction (STAT-3). The docking study confirms the affinity of Diosmin with apoptotic and important markers. Through the MTT assay, a dose-dependent cytotoxic effect of Diosmin was unveiled, with an IC50 value of effective inhibition of cell proliferation. Diosmin treatment resulted in noteworthy downregulation of Bcl-xl, Bak, Bcl-2, IL-1, 6, 8, TNF-α, and STAT-3 while upregulating the p53 and Bax expression levels, highlighting its inhibitory role in inducing apoptosis. Docking studies further exposed robust binding affinities between Diosmin and target apoptotic proteins, suggesting its efficacy in disrupting cellular functions and inflammatory signaling pathways in Hep-2 cells. The cytotoxic effects on Hep-2 cells and suggested activation of Bax, p53, and inhibition of Bcl-xl, Bak, Bcl-2, IL-1, 6, 8, TNF-α, as well as STAT-3 lead to the activation of mitochondrial-mediated apoptosis in Diosmin-treated Hep-2 cells. Further, its anti-inflammatory properties locate Diosmin as a conclusive compound for further studies for effective oral and other related squamous carcinoma treatments. © 2025 Elsevier B.V., All rights reserved.