The chytrid fungus Batrachochytrium dendrobatidis has been implicated as the causative agent of mass mortalities, population declines and the extinctions of amphibian species worldwide. Although several studies have shown that the prevalence of chytridiomycosis (the disease caused by the fungus) increases in cooler months, the magnitude and timing of these seasonal fluctuations have yet to be accurately quantified. We conducted disease sampling in a single population of stony creek frogs Litoria wilcoxii on 13 occasions over a 21-month period and used quantitative real-time polymerase chain reaction to detect and quantify the number of B. dendrobatidis zoospores present on samples. Disease prevalence varied significantly across sampling sessions, peaking at 58.3% (in early spring) and dropping to as low as 0% on two occasions (late summer and early autumn). There was a significant negative relationship between disease prevalence and mean air temperature in the 30 days prior to sampling. These large-scale seasonal fluctuations in chytridiomycosis levels will strongly influence conservation programs and amphibian disease research.