The antifungal activity of purpurin (1,2,4-trihydroxy-9,10-anthraquinone), a natural red anthraquinone pigment in madder root (Rubia tinctorum L.), was evaluated by a broth microdilution assay against a total of 24 Candida isolates representing six species. The minimum inhibitory concentration (MIC) range of purpurin was 1.28-5.12 μg/ml. Mechanistic studies using the rhodamine 6G extrusion assay indicated that purpurin inhibited the energy-dependent efflux pumps of the Candida isolates in a dose-dependent manner. Furthermore, purpurin demonstrated a dose-dependent depolarization of mitochondrial membrane potential, one of the biochemical checkpoints regulating cell death in eukaryotic cells, suggesting a possible linkage between purpurin antifungal mechanism and Candida apoptosis.