The objective of this study was to characterize the cutaneous antifungal bacterial flora of a plethodontid salamander, Plethodon cinereus, and to relate our results to aspects of amphibian ecology. Scanning electron micrographs of salamander skin indicated a wide distribution of bacteria across the skin. Polymerase Chain Reaction/Denaturant Gradient Gel Electrophoresis (PCR/DGGE) analyses of 16S rRNA gene fragments revealed that a transient community of bacteria was rinsed off and that a resident epibiotic community remained on the skin. Resident bacteria were isolated from the skin of P. cinereus on a low nutrient medium and challenged against a pathogenic ascomycete fungus collected from dead salamander eggs. The strong antifungal bacteria, identified by sequencing of the 16S rRNA gene (∼1400 bp), were related to the genera Lysobacter, Pseudomonas, Chryseobacterium, and Bacillus. Patterns of the cutaneous bacterial flora varied among individual salamanders, as revealed by DGGE, although analysis of DGGE bands from salamander skin revealed the presence of at least one species of uncultured β-proteobacteria on virtually every salamander. These bacteria were closely related to an isolated antifungal Janthinobacterium lividum strain. Antifungal skin bacteria may form mutualistic ecological relationships with amphibian species and help protect them from pathogenic fungi.