Oral cancer is a prevalent cancer in male worldwide. Oral potentially malignant disorders (OMPDs) are the oral mucosa lesions that have high malignant transformation rate to oral cancer. The mainstay for OMPDs treatment includes carbon dioxide (CO2) laser and surgery, which may lead to the side effects of scarring and impaired function of oral cavity in the patients and reduced their willingness to receive curative therapy. Therefore, developing a non-invasive and function-preserving therapy is clinically important. Since development of a novel chemotherapeutic drug requires a lot of time and cost, we applied the high-throughput screening (HTS) approach to identify new bioactivities for FDA-approved drugs, known as drug repurposing. Through this drug repurposing approach, we discovered that gentian violet (GV), which is well known for its antibacterial, antifungal, antihelminthic, antitrypanosomal and antiviral activities, was able to induce significant cell death in DOK oral precancerous cells through ROS production. Moreover, decreased phosphorylation of p53(Ser15) and NFκB(Ser536) was required for GV-induced cell death. In vivo, 3% GV orabase effectively suppressed the progression of DMBA-induced oral precancerous lesions. In conclusion, this new formulation of GV through drug repurposing has the potential to be further developed as a therapeutic drug for OPMD clinically.