Human oral squamous cell carcinoma (OSCC) represents a significant global health challenge, with conventional treatments showing limited efficacy in improving patient survival rates. To investigate the therapeutic potential of acetylshikonin on OSCC, we conducted comprehensive analyses including cell viability assays, flow cytometry, and molecular pathway investigations. Our findings demonstrate that acetylshikonin significantly inhibits OSCC cell proliferation with IC50 values of 3.81 μM and 5.87 μM in HSC3 and SCC4 cells respectively. Flow cytometry analysis revealed that acetylshikonin treatment significantly increased reactive oxygen species (ROS) production and decreased mitochondrial membrane potential in OSCC cells. Additionally, Western blot analysis showed enhanced phosphorylation of RIPK1, RIPK3, and MLKL proteins, indicating activation of the necroptotic pathway. The critical role of necroptosis was further confirmed using specific inhibitors (GSK872, Necrostatin-1, and 7-CL-O Nec-1), which significantly attenuated acetylshikonin-induced cell death. Transmission electron microscopy revealed distinct ultrastructural changes in cellular organelles, while decreased GPX4 expression suggested potential cross-activation of ferroptotic pathways. These data demonstrate that acetylshikonin suppresses OSCC growth through selective activation of oxidative stress-mediated necroptosis and mitochondrial dysfunction, identifying it as a promising natural compound for OSCC therapy through its ability to activate alternative cell death pathways and overcome traditional therapy limitations.