The Buffalo National River in Arkansas was established as the first national river in 1972 and later designated as an Extraordinary Resource Water with a Wild and Scenic section in the upper portion. The Buffalo National River's water quality database indicates nitrogen compounds have increased since the 1980s. This study aimed to investigate the potential threat of elevated nutrients and its association with freshwater mussel declines in the Buffalo National River through laboratory and in situ exposures. We evaluated (1) the toxicity of receiving water and sediment samples containing elevated ammonia and nitrate to native juvenile Lampsilis reeveiana (Arkansas Brokenray) and three common aquatic test species (Ceriodaphnia dubia, Pimephales promelas, and Hyallela azteca) and (2) in situ bioassays with juvenile L. reeveiana to investigate the effects of nitrogen contaminants and other water quality factors on survival and growth.