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Glucansucrase secreted by <i>Streptococcus mutans</i> 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 <i>S. mutans</i> and its enzyme glucansucrase implicated in biofilm formation. Antibacterial activity against planktonic <i>S. mutans</i> was carried out. Using reverse transcription PCR, the expression of crucial virulence genes, <i>gtfB</i>, <i>gtfC</i>, <i>gtfD</i>, <i>gbpB</i>, and <i>comDE</i> were determined. The effect of mangiferin on teeth surfaces biofilm was ascertained by scanning electron microscopy (SEM). Docking analysis of <i>S. mutans</i> glucansucrase and mangiferin revealed the binding energy of −7.35 and ten hydrogen interactions. Antibacterial study revealed that mangiferin was not lethal to planktonic <i>S. mutans,</i> 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 <i>S. mutans</i> biofilm density was scanty in mangiferin treated teeth compared to non-treated control teeth. Our data therefore suggest that mangiferin inhibited <i>S. mutans</i> biofilms formation by attenuating glucansucrase activities without affecting bacteria growth.