Objective: Betel quid (BQ) chewing habit is a risk factor of oral cancer and oral submucous fibrosis (OSF). Safrole is one of the major polyphenolic compounds in the inflorescence of Piper betle. However, little is known about the pathobiological effects of safrole in human buccal mucosal fibroblasts (BMFs). Method: In this study, human BMFs were derived from three healthy patients with the informed consent. The effects of safrole on fibroblasts were investigated by cytotoxicity, attachment and migration assay. In addition, the effects of antioxidants, superoxide dismutase (SOD, a superoxide scavenger), catalase (a hydrogen peroxide scavenger), 2-oxothiazolidine-4-carboxylic acid (OTZ, a glutathione synthesis precursor) were added to find their possible mechanisms of action, as well as a method for the prevention of BQ chewing-associated OSF. Results: Our study exhibited that safrole was cytotoxic to BMFs in a dose-dependent manner (p<0.05). Safrole also inhibited cell migration and attachment in a dose- and time-dependent manner. Moreover, the addition of SOD and catalase, but not OTZ acted as a protective effect on the safrole-induced cytotoxicity. At the concentration of 1µg/ml safrole, cell viability was 67%. SOD and catalase increased cell viability upto 93% and 90% at the concentration of 1 µg/ml safrole, respectively (p<0.05). Conclusion: Our results indicate that safrole take part in the pathobiological effects of BQ chewing-associated OSF. Besides, the use of free radical scavengers may provide further chemoprevention of BQ chewing-associated OSF.