Agricultural practices pose threats to biotic diversity in freshwater systems with increasing use of glyphosate-based herbicides for weed control and animal waste for soil amendment becoming common in many regions. Over the past two decades, these particular agricultural trends have corresponded with marked declines in populations of fish and mussel species in the Upper Conasauga River watershed in Georgia/Tennessee, USA. To investigate the potential role of agriculture in the population declines, surface waters and sediments throughout the basin were tested for toxicity and analyzed for glyphosate, metals, nutrients, and steroid hormones. Assessments of chronic toxicity with Ceriodaphnia dubia and Hyalella azteca indicated that few water or sediment samples were harmful and metal concentrations were generally below impairment levels. Glyphosate was not observed in surface waters, although its primary degradation product, aminomethyl phosphonic acid (AMPA), was detected in 77% of the samples (mean = 509 μg/L, n = 99) and one or both compounds were measured in most sediment samples. Waterborne AMPA concentrations supported an inference that surfactants associated with glyphosate may be present at levels sufficient to affect early life stages of mussels. Nutrient enrichment of surface waters was widespread with nitrate (mean = 0.7 mg NO₃-N/L, n = 179) and phosphorus (mean = 275 μg/L, n = 179) exceeding levels associated with eutrophication. Hormone concentrations in sediments were often above those shown to cause endocrine disruption in fish and appear to reflect the widespread application of poultry litter and manure. Observed species declines may be at least partially due to hormones, although excess nutrients and herbicide surfactants may also be implicated.