Considering the importance of benthic macroinvertebrates for diagnosis of variations in the ecological conditions of aquatic habitats, the aim of this study was to investigate the structure of the Chironomidae and Oligochaeta assemblages along an organic pollution gradient. The fauna specimens were obtained with the use of artificial substrates, and the environmental variables were recorded at five sites of the São Lourenço River, during 12 months. Metrics of the assemblage and detrended correspondence analysis were used to verify the response of the fauna to the pollution gradient. Procrustes analysis was used to verify whether the data on the Chironomidae and Oligochaeta assemblages, as well as the taxonomic and numerical resolution of these groups, provide similar results in relation to the pollution gradient. The richness, evenness, and taxonomic composition of the Chironomidae and Oligochaeta assemblages varied significantly among the collection sites, with distinct conservation conditions. Genera of the subfamilies Orthocladiinae and Tanypodinae were associated with the sites upstream of the urban area, where the dissolved oxygen levels are higher. Species of Oligochaeta and the genus Chironomus were associated with more organically polluted sites. No concordance was observed in the response of the Chironomidae and Oligochaeta assemblages in relation to the environmental variables, indicating the need to use both groups in biomonitoring studies. On the other hand, both the data on composition (presence or absence) and those on the lowest taxonomic resolution (abundance of subfamilies) were effective to diagnose the pollution gradient in the river studied. Therefore, when the environmental conditions along a river's gradient are contrasting, we suggest the use of the lowest taxonomic resolution of Chironomidae and Oligochaeta in biomonitoring. That procedure considerably reduces the assessment time, besides being a method that can be used by people not specializing in the taxonomy of groups.