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Glucansucrase secreted by Streptococcus mutans and composed of virulence genes alters oral microbiota, creating adherent environment for structural bacteria colony forming dental biofilm. The present investigation studied the inhibitory and binding potentials of mangiferin against S. mutans and its enzyme glucansucrase implicated in biofilm formation. Antibacterial activity against planktonic S. mutans was carried out. Using reverse transcription PCR, the expression of crucial virulence genes, gtfB, gtfC, gtfD, gbpB, and comDE were determined. The effect of mangiferin on teeth surfaces biofilm was ascertained by scanning electron microscopy (SEM). Docking analysis of S. mutans glucansucrase and mangiferin revealed the binding energy of −7.35 and ten hydrogen interactions. Antibacterial study revealed that mangiferin was not lethal to planktonic S. mutans, but a concentration-dependent inhibition of glucansucrase activity was observed. The inhibitory effect of water-insoluble glucan synthesis was apparently more marked relative to water-soluble glucan synthesis attenuation. Mangiferin significantly downregulated the expression of the virulence genes, indicating a mechanism involving glucanotranferases, specifically inhibiting colony formation by attenuating bacterial adherence. SEM images revealed that S. mutans biofilm density was scanty in mangiferin treated teeth compared to non-treated control teeth. Our data therefore suggest that mangiferin inhibited S. mutans biofilms formation by attenuating glucansucrase activities without affecting bacteria growth.