Amphibian declines and extinctions have been documented around the world, often in protected natural areas. Concern for this alarming trend has prompted the U.S. Geological Survey and the National Park Service to document all species of amphibians that occur within U.S. National Parks and to search for any signs that amphibians may be declining. This study, an inventory of amphibian species in Biscayne National Park, was conducted during 2002 and 2003. The goals of the project were to create a georeferenced inventory of amphibian species, use new analytical techniques to estimate proportion of sites occupied by each species, look for evidence of known stressors or problems that may lead to amphibian population decline (invasive species, disease, die-offs, and so forth), and to establish a baseline and methodology that could be used for future monitoring efforts. Four sampling methods were used to accomplish these goals. Visual encounter surveys and anuran vocalization surveys were conducted at a total of 236 visits to 37 sites in all habitats throughout Biscayne National Park to estimate the proportion of sites or proportion of area occupied (PAO) by each amphibian species in each habitat. More than 100 individuals of 7 amphibian species were detected during standard sampling, and 24 individuals of 6 species of amphibians and 37 individuals of 12 species of reptiles were encountered during opportunistic collections and nighttime road surveys used to augment the visual encounter methods for more rare or cryptic species opportunistically. The software PRESENCE was used to provide PAO estimates for each of the anuran species based on the visual encounter surveys and anuran vocalization data. Amphibian species (six native and three non-native) were documented in Biscayne National Park during this project. The proportion of area occupied estimates obtained for the six most common amphibians will serve as a comparative baseline for future monitoring efforts. There were fourteen non-marine reptile species detected during this study. The proportion of area occupied for reptile species was not estimated because there were too few encounters during this study. The methods used in this study are adequate to produce reliable estimates of the proportion of sites occupied by most anuran species. Therefore, future sampling at regular intervals could be a cost-effective way of following amphibian occupancy trends. This study identified some threats to amphibians in Biscayne National Park, especially introduced species including the Cuban treefrog (Osteopilus septentrionalis), the marine or cane toad (Bufo marinus), and the greenhouse frog (Eleutherodactylus planirostris planirostris) that were collectively detected nearly three times as often as native species.