Usnic acid (UA) is a unique bioactive substance in lichen with potential anticancer properties. Recently, we have reported that UA can reduce 7,12‐dimethylbenz[a] anthracene‐induced oral carcinogenesis by inhibiting oxidative stress, inflammation, and cell proliferation in a male golden Syrian hamster in vivo model. The present study aims to explore the relevant mechanism of cell death induced by UA on human oral carcinoma (KB) cell line in an in vitro model. We found that UA can induce apoptosis (cell death) in KB cells by decreasing cell viability, increasing the production of reactive oxygen species (ROS), depolarizing mitochondrial membrane potential (MMP) levels, causing nuclear fragmentation, altering apoptotic morphology, and causing excessive DNA damage. Additionally, UA inhibits the expression of Bcl‐2, a protein that promotes cell survival, while increasing the expression of p53, Bax, Cytochrome‐c, Caspase‐9, and 3 proteins in KB cells. UA also inhibits the expression of nuclear factor‐κB (NF‐κB), a protein that mediates the activation of pro‐inflammatory cytokines such as TNF‐α and IL‐6, in KB cells. Furthermore, UA promotes apoptosis by enhancing the mitochondrial‐mediated apoptotic mechanism through oxidative stress, depletion of cellular antioxidants, and an inflammatory response. Ultimately, the findings of this study suggest that UA may have potential as an anticancer therapeutic agent for oral cancer treatments.