Macrobenthic communities from estuaries throughout the northern Gulf of Mexico were studied to assess the influence of sediment contaminants and natural environmental factors on macrobenthic community trophic structure. Community trophic data were also used to evaluate whether results from laboratory sediment toxicity tests were effective indicators of site-specific differences in benthic trophic structure. A multiple regression model consisting of five composite factors (principal components) was used to distinguish the effects of sediment contaminants and environmental variables on benthic community trophic structure. This model explained 33.5% of the variation in macrobenthic trophic diversity (p<0.001), a variable derived from the distribution of taxas among nine original trophic categories. A significant negative relatinship was found between principal components reflecting concentrations of sediment contaminants and macrobenthic trophic diversity. Detritivores including surface deposit-feeders (SDF), subsurface deposit-feeders (SSDF), and filter feeders (FF) were numerically dominant at 201 random sites, each group accounting for 25–30% of total macrobenthic abundance. The relative abundance of SDFs was considerably lower (12.1±2.9% to 17.1±4.4%) at sites where sediment contaminant concentrations exceeded minimum biological effects thresholds (ER-L values from Long and Morgan 1990 than at sites sampled at random (29.3±5.7%). SSDFs were proportionally more abundant at contaminated sites (42.0±7.7% to 63.6±10.3%) versus random sites (27.5±5.7%), and the relative abundance of SSDFs was positively correlated with concentrations of particular contaminants. Benthic trophic structure was also found to be a function of salinity, where the proportion of SSDFs was negatively correlated with salinity (p=0.035, r=−0.223, n=326). Silt-clay content loaded fairly strongly on the first principal component, but trophic structure parameters were not significantly correlated with sediment grain size or dissolved oxygen (perhaps due, in part, to covariation). Results from laboratory sediment toxicity tests with mysids were predictive of differences in macrobenthic trophic structure in situ (i.e., mysid survival was negatively correlated with %SSDF; p<0.001, r=−0.292, n=326). Results from laboratory sediment toxicity tests with ampeliscid amphipods were not indicative of site-specific differences in benthic trophic structure. Results from this study demonstrated that sediment contaminants can be quite important in structuring macrobenthic communities in soft-bottom estuarine habitats. The fact that macrobenthic trophic diversity decreased significantly with increasing sediment contamination indicates that important general differences in benthic community function may exist between contaminated and random sites. These data suggest that benthic trophic structure analysis may be an effective tool for assessing integrated community responses to chronic sublethal exposure and may be useful for assessing toxicological responses at ecologically relevant levels of organization.