Background: Thymoquinone (TQ), an active component of Nigella sativa, has been shown to reduce tumor growth in oral cancer models. However, toxicity at high doses with poor water solubility limits its usage as a therapeutic agent. Nanoformulations were introduced to overcome these problems. Aim: The present study was designed to evaluate the effect of different nanothymoquinone (NTQ) concentrations on the chemically-induced epithelial dysplasia of the hamster buccal pouch. Materials and Methods: NTQ was prepared to improve the effectiveness of TQ. Transmission electron microscopy confirmed the particle diameter between 6 to 15 nm. Clinical observations, histopathological alterations and complete blood count were used to investigate the effect of TQ and NTQ on severe oral epithelial dysplasia induced on hamsters' buccal pouches by 7,12-dimethylbenz[a]anthracene (DMBA). Animals received DMBA, three times weekly for six weeks then were treated with intraperitoneal injection of TQ (0.1 mg/kg) or different concentrations of NTQ (0.1, 0.05, 0.025 and 0.01 mg/kg) three times per week for another 6 weeks. Results: TQand higher concentrations of NTQhad higher anti-tumor activity towards the severe epithelial dysplasia. The best result was with NTQ 0.1mg/kg concentration. Conclusion: NTQ is a promising candidate in cancer chemoprevention and chemotherapy as well as striated muscle regeneration.