A tea tree oil (TTO) preparation of defined chemical composition was studied, using a microbroth method, for its in vitro activity against 115 isolates of Candida albicans, other Candida species and Cryptococcus neoformans. The fungal strains were from HIV-seropositive subjects, or from an established type collection, including reference and quality control strains. Fourteen strains of C. albicans resistant to fluconazole and/or itraconazole were also assessed. The same preparation was also tested in an experimental vaginal infection using fluconazole-itraconazole-susceptible or -resistant strains of C. albicans. TTO was shown to be active in vitro against all tested strains, with MICs ranging from 0.03% (for C. neoformans) to 0.25% (for some strains of C. albicans and other Candida species). Fluconazole- and/or itraconazole-resistant C. albicans isolates had TTO MIC50s and MIC90s of 0.25% and 0.5%, respectively. TTO was highly efficacious in accelerating C. albicans clearance from experimentally infected rat vagina. Three post-challenge doses of TTO (5%) brought about resolution of infection regardless of whether the infecting C. albicans strain was susceptible or resistant to fluconazole. Overall, the use of a reliable animal model of infection has confirmed and extended our data on the therapeutic effectiveness of TTO against fungi, in particular against C. albicans.