The real-time PCR was performed to assess S. mutans virulence genes. The biofilms treated with Nystatin had down-regulations of gtfB, and eno genes, compared to the control group, despite no statistically significant differences were detected. The expression of S. mutans gtfD and atpD was significantly down-regulated (p<0.05, t-test) in the Nystatin-treated biofilms than the control group at 44 hours when 4 Nystatin applications had been administered (p<0.05, t-test)