The Piratinim River is located in the northwest of Rio Grande do Sul, and represents an important effluent from the Uruguay River, with streams located far from urbanized areas, in conditions similar to those of environmental integrity, but under the influence of agricultural activities. In this study, we aim at investigating the structure of the aquatic macroinvertebrate community in streams of the Piratinim river basin by observing both spatial and local scales. The sampling was carried out in six streams distributed in three regions (upper, middle and lower) of the basin, thus exploring its upstream, intermediate and downstream stretches, during spring and autumn. Macroinvertebrates were collected using aquatic dipnets and were identified at the family level; trophic-functional categories were established according to the classification adapted to the state of Paraná. The spatial and temporal variations of the groups and of the biotic attributes (density, richness, diversity and equitability) were subjected to Kruskal-Wallis non-parametric tests and a posteriori Dunn’s tests. The abiotic variables were obtained to verify possible influence on the composition of the macroinvertebrate community, evaluated through a Canonical Correspondence Analysis. We sampled 11,564 macroinvertebrate individuals from 72 taxa, and found a predominance of the collector-filter trophic group. Abundance and richness were different between streams; the highest densities were found in the streams located in the upper region of the watershed (Chuní and Itú). The highest taxon richness was found in the lower region of the watershed (Guaracapa stream), and the lowest richness was found in the two streams for the intermediate region (Santana and Ximbocu). Diversity and equitability did not vary; temporal variations were not found. Canonical correspondence analysis explained 31.7% of the data variability. The main environmental variables that influenced macroinvertebrates distribution were temperature, electrical conductivity, dissolved oxygen, altitude and extension of the riparian forest. Seasonality and the longitudinal gradient along the basin represented determining factors for the structure and distribution of the macroinvertebrate community in the tributary streams of the Piratinim River.