Beneficial bacteria that live on salamander skins have the ability to inhibit pathogenic fungi. Our study aimed to identify the specific chemical agent(s) of this process and asked if any of the antifungal compounds known to operate in analogous plant-bacteria-fungi systems were present. Crude extracts of bacteria isolated from salamander skin were exposed to HPLC, UV-Vis, GC-MS, and HR-MS analyses. These investigations show that 2,4-diacetylphloroglucinol is produced by the bacteria isolate Lysobacter gummosus (AB161361), which was found on the red-backed salamander, Plethodon cinereus. Furthermore, exposure of the amphibian fungal pathogen, Batrachochytrium dendrobatidis (isolate JEL 215), to different concentrations of 2,4-diacetylphloroglucinol resulted in an IC50 value of 8.73 microM, comparable to crude extract concentrations. This study is the first to show that an epibiotic bacterium on an amphibian species produces a chemical that inhibits pathogenic fungi.