In the present study, we investigated the anticarcinogenic potential of Boldine, a natural alkaloid, against human oral carcinoma KB and HEp-2 cell lines. Cytotoxicity was evaluated using the MTT assay, while the antioxidant and cytoprotective effects were assessed by measuring the reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) through DCFH-DA staining and Rhodamine-123, respectively. DAPI and AO/EtBr staining analyzed nuclear damage and apoptotic morphological alterations. Cells treated with Boldine demonstrated decreased viability, elevated ROS levels, disrupted mitochondrial membrane potential, significant nuclear fragmentation, and DNA damage. Furthermore, Boldine influenced apoptotic markers Bax, Cytochrome c, and caspases 3 and 9 is upregulating expression while Bcl-2 biomarker is downregulating. It also affected the NOTCH signaling by modifying the expression of Notch1, Hes1, Hey1, and Jagged1. This study underscores the role of Boldine in interfering with critical pathways related to cell survival and apoptosis regulation. Its capacity to increase oxidative stress and modulate mitochondrial function further emphasizes its therapeutic potential. By targeting both intrinsic and extrinsic apoptotic pathways, Boldine effectively inhibits tumor growth. These results position Boldine as a promising candidate for developing anticancer therapies aimed at aggressive malignancies such as KB and HEp-2 cells. © 2025 Elsevier B.V., All rights reserved.