Our previous study showed that concomitant use of berberine (BBR) and fluconazole (FLC) provided a synergistic action against FLC-resistant Candida albicans (C. albicans) clinical strains in vitro. To clarify the mechanism underlying this action, we performed a comparative proteomic study in untreated control cells and cells treated with FLC and/or BBR in 2 clinical strains of C. albicans resistant to FLC. Our analyses identified 16 differentially expressed proteins, most of which were related to energy metabolisms (e.g., Gap1, Adh1, and Aco1). Functional analyses revealed that FLC + BBR treatment increased mitochondrial membrane potential, decreased intracellular ATP level, inhibited ATP-synthase activity, and increased generation of endogenous reactive oxygen species (ROS) in FLC-resistant strains. In addition, checkerboard microdilution assay showed that addition of antioxidant ascorbic acid or reduced glutathione reduced the synergistic antifungal activity of FLC + BBR significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the synergistic action of FLC + BBR against FLC-resistant C. albicans.