A concentrated animal feeding operation (CAFO) established in Newton County, Arkansas, near Big Creek, a tributary of the Buffalo National River, raised concern about potential degradation of water quality in the karst watershed. In this study, isotopic tools were combined with standard geochemical approaches to characterize nutrient sources and dynamics in the Big Creek watershed. An isotopic and geochemical reference database of potential nutrient sources in the Big Creek watershed was constructed based on samples collected from representative potential sources. Nutrient sources and stream samples were analyzed for delta (δ)¹⁵N-NO₃, δ¹⁸O NO₃, and a suite of selected dissolved ions. Data provide evidence of modification of potential local nutrient source signatures by nitrification, atmospheric deposition, evaporation, and denitrification. Samples taken from the CAFO waste pond, a septic system, field and parking lot runoff, fertilizer, and hog manure exhibited different δ¹⁵N-NO₃ and δ¹⁸O-NO₃ values as compared to stream samples. Stream δ¹⁵N-NO₃ and δ¹⁸O-NO₃ values cannot be explained by direct input of any one of these potential sources without modification of the isotopic composition by mixing or fractionation. Big Creek nitrate isotope values (-3.4 per mil [‰] to 6.7‰ δ¹⁵N-NO₃ and -7.6 to 9.1‰ δ¹⁸O-NO₃) were similar to values expected from nitrification of nitrogen stored in soils sampled in the watershed (2.8 to 7.6‰ δ¹⁵N-NO₃ and 3.4 to 4.8‰ δ¹⁸O-NO₃).