Researchers have placed emphasis on quantifying and identifying ecological thresholds to study biological responses to urbanization. As watersheds become urbanized, they exhibit a systemic pattern of degradation that disrupts the natural biogeochemical and geomorphologic processes, ultimately leading to a decline in freshwater biodiversity. In North Carolina, an increase in population is leading to an aquatic biodiversity crisis which can be observed in declining freshwater fish abundance and diversity. Although studies in the Eastern Piedmont and specifically North Carolina have quantified the relationship between aquatic biotic communities and urbanization, they have fallen short of identifying individual tolerances. This study uses land cover data from the National Land Cover Database and biomonitoring datasets from the North Carolina Department of Environmental Quality and Mecklenburg County Stormwater Services with the Threshold Indicator Taxa Analysis (TITAN) to quantify percent impervious cover (IC) thresholds and change points at the community and individual level to identify biological indicators and conservation priorities for watershed health. Non-parametric and pairwise testing was used to identify IC tolerance trends among ecological functional groups and pollution tolerance designations. Results of the land cover analysis reveal that IC increased by 1.73% throughout the North Carolina Piedmont in the 16-year period, but watersheds with < 15% IC decreased by 9.7% and watersheds between 45-60% increased 329%. TITAN revealed that Z- taxa experience the greatest change in frequency and abundance, also known as change point, at 6.10% IC and have an aggregate threshold of ~ 7% IC (5.79-12.78%); Z+ taxon have a change point of 16.59% and an aggregate threshold of 41.30% IC (16.07-57.37%). Kruskal-Wallis results demonstrated IC tolerance among pollution tolerance classifications and trophic guilds thresholds were significant but insignificant for spawning guilds. TITAN also revealed several taxa whose IC tolerances differentiated from their respected pollution tolerance. Overall, this study revealed that with the current NC state watershed development regulations, ~88% of the state’s watersheds could exceed IC thresholds of ~75% of taxa within the NCP fish assemblage.