Spatial variation in the density and biomass of Branchiostoma caribaeum was analyzed along a sewage contamination gradient identified by fecal steroids in a subtropical estuary, southern Brazil. Sampling, repeated in the austral winter and summer, followed a hierarchical design nested at four spatial scales (sector>1km; area>100m; site>10m; replicate<1m). Density and biomass were significantly lower at sites characterized by high concentrations of fecal steroids. The best combinations of variables that explained the biological similarities among sites involved contamination indicators. Most of the variation of biological data was found at the smallest scales and could be related with the sediment texture. Our study highlighted the usefulness of a multi-scale perspective to evaluate distribution patterns of benthic invertebrates as a biological indication of environmental pollution. Gradient analyses at larger spatial scales may be invalidated by the patchy distribution of benthic fauna if they do not account for such small scale variability.