The northern Gulf of Mexico (GoM) spans five U.S. states and encompasses estuaries that vary greatly in size, shape, upstream river input, eutrophication status, and biotic communities. Given the variability among these estuaries, assessing their biological condition relative to anthropogenic stressors is challenging, but important to regional fisheries management and habitat conservation initiatives. Here, a hierarchical generalized linear modeling approach was developed to predict species presence in bottom trawl samples, using data from 33 estuaries over a nineteen-year study period. This is the first GoM estuary assessment to leverage Gulf-wide trawl data to develop species-level indicators and a quantitative index of estuary disturbance. After controlling for sources of variability at the sampling event, estuary, state, and sampling program levels, our approach screened for statistically significant relationships between watershed-level anthropogenic stressors and fish and invertebrate species presence. Modeling results indicate species level indicators with sensitivities to landscape stressor gradients. The most influential stressors include total anthropogenic land use, crop land use, and the number of toxic release sites in upstream watersheds, as well as agriculture in the shoreline buffer, each of which was significantly related to between 21% and 39% of the 57 species studied. Averaging the effects of these influential stressors across species, we develop a quantitative estuary stress index that can be compared against benchmark conditions. In general, disturbance levels were greatest in estuaries west of the Mississippi delta and in highly developed estuaries in southwest Florida. Estuaries from the Florida panhandle to the eastern Mississippi delta had less anthropogenic stress.