To detect aquatic animal diseases of national concern, 111 individual amphibians, including wood frogs Rana sylvatica (28), spring peepers Pseudacris crucifer (35), red-spotted newts Notophthalmus viridescens (41), and gray tree frogs Hyla versicolor (7), were sampled at seven different sites in the Delaware Water Gap National Recreation Area (DGNRA), Pennsylvania, from June 14 to July 19, 2007. These samples were screened for Batrachochytrium dendrobatidis and viral pathogens at the U.S. Fish and Wildlife Service's Fish Health Center in Lamar, Pennsylvania. Cell culture revealed cytopathic effect (CPE) in two cell lines (epithelioma papillosum cyprini and fathead minnow) inoculated with liver, kidney, and spleen samples from one sample pool of Notophthalmus viridescens (4 individuals). Polymerase chain reaction was conducted on cell culture supernatant exhibiting CPE. Sequencing revealed the resulting product to be identical to frog virus 3, a ranavirus in the family Iridoviridae. Upon gross examination, two Notophthalmus viridescens were found to exhibit dermal swelling and lethargy. Histological examination of these lesions revealed involvement by an Ichthyophonus sp. In summary, two pathogens of concern were found in amphibians in the DGNRA: a ranavirus with a major capsid protein sequence identical to that of frog virus 3 and a mesomycetozoan, Ichthyophonus sp. Although no epizootic die-offs were observed during this health survey, the results warrant further research into the distribution of these pathogens throughout the DGNRA because they have the potential to cause mass mortalities in amphibians.