Objectives: The aim of this study was to investigate the mechanism of action of tea tree oil and its components against Candida albicans, Candida glabrata and Saccharomyces cerevisiae.Methods: Yeast cells were treated with tea tree oil or components, at one or more concentrations, for up to 6 h. During this time, alterations in permeability were assessed by measuring the leakage of 260 nm absorbing materials and by the uptake of Methylene Blue dye. Membrane fluidity was measured by 1,6-diphenyl-1,3,5-hexatriene fluorescence. The effects of tea tree oil on glucose-induced medium acidification were quantified by measuring the pH of cell suspensions in the presence of both tea tree oil and glucose.Results: The treatment of C. albicans with tea tree oil and components at concentrations of between 0.25 and 1.0% (v/v) altered both permeability and membrane fluidity. Membrane fluidity was also increased when C. albicans was cultured for 24 h with 0.016%-0.06% (v/v) tea tree oil, as compared with control cells. For all three organisms, glucose-induced acidification of the external medium was inhibited in a dose-dependent manner in the presence of 0.2%, 0.3% and 0.4% tea tree oil.Conclusions: Data from this study support the hypothesis that tea tree oil and components exert their antifungal actions by altering membrane properties and compromising membrane-associated functions.