Background: Tooth decay is one of the most common health problems in the world. Nowadays, finding new compounds to prevent tooth decay is more necessary than ever. The purpose of this study was to compare the effects of fluoride 0.2% and mouthwash combined with flavonoid compounds against bacterial samples. Methods: The crystal structures of glucansucrase from Streptococcus mutans and glucan-1, 6-alpha-glucosidase from Lactobacillus acidophilus were obtained from the Protein Data Bank. By using AutoDockTools (1.5.7), ligands and protein interactions were calculated and ready for AutoDock vina. The agar well diffusion and the minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) methods were used to investigate the inhibitory effect of mouthwashes, and the results were obtained with SPSS software. Results: Compounds eriocitrin and galangin showed the highest amount of H-bonds with amino acids against glucansucrase. In addition, catechin, eriocitrin, and isorhamnetin compounds demonstrated the highest amount of H-bonds with amino acids against glucan-1, 6-alpha-glucosidase. In vitro results revealed that groups a (Fluoride 0.2%+eriocitrin against S. mutans) and d (Fluoride 0.2%+eriocitrin against L. acidophilus) represented the most effect among all compounds, respectively (Inhibition zone=26±0.5 mm, MIC=250 µg/mL, MBC=500 µg/mL and inhibition zone=31±0.5 mm, MIC=125 µg/mL, MBC=250 µg/mL). Conclusion: Fluoride 0.2% with eriocitrin was more effective in both methods (In silico and in vitro) compared to fluoride 0.2% due to its good inhibitory effect at different concentrations against S. mutans and L. acidophilus.