Eight-ring hairpin polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids have been shown to bind with subnanomolar affinity to discrete DNA sites and to modulate a variety of DNA-dependent biological processes. We show here that addition of a second positive charge at the C terminus of an 8-ring hairpin polyamide confers activity against a number of clinically relevant fungal strains in vitro, and activity against Candida albicans in a mouse model. Control experiments indicate that the observed antifungal activity results from a DNA binding mechanism-of-action that does not involve DNA damage or disruption of chromosomal integrity. Hairpin activity is shown to be proportional to yeast DNA content (ploidy). Transcriptional interference is proposed as the likely explanation for fungal cytotoxicity. Experiments with sensitized yeast strains indicate the potential for discrete sites of action rather than global effects.