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This work was aimed at purifying the most potent antibiofilm principle from Acalypha wilkesiana leaves against Candida species, analyzing its binding interactions with the molecular targets and evaluating its mode of action. Bioassay guided fractionation was carried and purification of the most potent fraction was achieved by Preparative -TLC. Proton NMR -spectroscopy was used to elucidate the structure of the most potent fraction which was hydroxy tyrosol (HT). There was significant (p < 0.05) increase in the IC50 of HT and caspafungin in the presence of sorbitol. In the presence of ergosterol, there was no significant (p > 0.05) increase in the IC50 of HT but there was significant (p < 0.05) increase in the IC50 of voriconazole. Insilico molecular studies revealed a good docking score (-7.7 and 4 hydogen bonds) with glucan synthase and (7.0 and 1 hydrogen bond) with lanosterole-14a-demethylase. The mode of action of HT is most likely by inhibiting the activities of ß-1,3-D glucan synthase. The Significant increase in IC50 of HT in the presence of sorbitol showed that its inhibition leads to depletion of cell wall glucan and subsequent lysis of fungal cells. [ABSTRACT FROM AUTHOR]