Subsidy–stress gradients offer a useful framework for understanding ecological responses to perturbation and may help inform ecological metrics in highly modified systems. Historic, region-wide shifts from bottomland hardwood forest to agriculture can cause positively skewed gradients in alluvial plain ecoregions, resulting in tolerant organisms that exhibit a subsidy response (increased abundance in response to environmental stressors) shifting to a stress response (declining abundance at higher concentrations). Thus, observed biological tolerance in modified ecosystems may differ from less modified regions, creating challenges for detecting responses to restoration. Using the agriculturally dominated Mississippi Alluvial Plain (MAP) ecoregion in Mississippi, USA, as a case study, we hypothesized that macroinvertebrate taxa that typically display a subsidy response to nutrient enrichment in less modified ecoregions (i.e., nutrient-tolerance) shift to a stress response to increasing nutrients in highly modified watersheds with elevated nutrient conditions (i.e., nutrient intolerance). The abundance and diversity of MAP-specific intolerant taxa identified with threshold indicator taxa analysis were either unresponsive or exhibited a subsidy response to increasing nutrients in less modified ecoregions in Mississippi with lower nutrient concentrations, but declined at higher concentrations, providing evidence for a stress response to elevated nutrients in the MAP. Additionally, MAP-specific tolerant and intolerant taxa richness responded to increased nutrients predictably and consistently across space and time within the MAP. However, in MAP streams, elevated specific conductance was predicted to dampen the response of tolerant and intolerant taxa richness to increasing nutrient concentrations, highlighting the importance of considering multi-stressor interactions when interpreting biological data. Lastly, we demonstrate the efficacy of this approach with sediment bacterial communities characterized with amplicon sequencing, which lack sufficient life history characteristics necessary for the development of multi-metric indices. Both macroinvertebrate and bacterial communities responded similarly to increasing nutrient concentrations, suggesting DNA-based approaches may provide an efficient biological assessment tool for monitoring water quality improvements in highly modified watersheds.