In light of the need for new antifungal regimens, we report that at noncandidacidal concentrations, the lactoferrin-derived peptide hLF(1-11), which is highly active against fluconazole-resistant Candida albicans, acts synergistically with fluconazole against this yeast and a fluconazole-sensitive C. albicans strain as well as C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. When these yeasts were exposed to hLF(1-11) for 5 min and then incubated with fluconazole, they were killed effectively, while no candidacidal activity was observed when they were incubated first with fluconazole and then exposed to the peptide, indicating that the candidacidal activity is initiated by the peptide while fluconazole is only required during the effector phase. Investigations of the effect of azide, which inhibits mitochondrial respiration, on the activity of combinations of hLF(1-11) and fluconazole against fluconazole-resistant C. albicans revealed that it inhibits this activity, even when added during the effector phase only. As expected, azide inhibited the accumulation of rhodamine 123 in mitochondria and the production and release of ATP by C. albicans that occurred upon exposure to the combination of hLF(1-11) and fluconazole. Accordingly, oxidized ATP (oATP), an antagonist of ATP receptors, completely blocked the candidacidal activity of the hLF(1-11)-fluconazole combination, whereas oATP did not block the activity when its presence was restricted to the effector phase. The candidacidal activity of combinations of hLF(1-11) and fluconazole, which is initiated by the peptide through the involvement of energized mitochondria, renders fluconazole-resistant C. albicans sensitive to this azole.