Many amphibian species are threatened with extinction by the emerging infectious disease, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis. This unprecedented global crisis threatens to reduce the biodiversity of the entire amphibian class. The fungus invades the skin and impairs the uptake and retention of essential ions leading to cardiac arrest. Antimicrobial peptides (AMPs) secreted into the mucus of some amphibians are thought to be an important defense against chytridiomycosis. However, little is known about the quantities of AMPs secreted under natural conditions, whether they are sufficient to protect against this pathogen, and how they interact with commensal microbes. To understand how defensive peptides and skin microbes may interact, it is essential to know the precise quantities of AMPs present under natural conditions. Using matrix-assisted laser desorption time-of-flight mass spectrometry and growth inhibition assays, we show that northern leopard frogs (Rana pipiens) at rest constitutively release low amounts of AMPs that inhibit B. dendrobatidis in vitro, and AMP defenses are elevated following a simulated predator attack. Using a synthetic peptide analogue of brevinin-1Pb as an external control, we quantified the amounts of four previously described AMPs (brevinin 1Pa, brevinin-1Pb, brevinin-1Pd, and ranatuerin-2P) at several time points after secretion. Once secreted onto the skin, the peptides are most active for 15 min, and small quantities persist for at least 2 h. Taken together, our data suggest that small amounts of AMPs are rapidly available and quite stable on the skin of R. pipiens. They are effective inhibitors of B. dendrobatidis at these low constitutive concentrations but degrade within 2 h, protecting the integrity of the skin and commensal bacteria.