The rectified channel was installed in the Itajaí-Mirim river in the mid-1960s to increase the river flow in the urban area of Itajaí, which suffers from flooding and inundation during periods of high precipitation. The present study aimed to evaluate the spatio-temporal variations of the benthic macrofauna of the Itajaí-Mirim river, in the original and rectified channels. The distribution of organisms was evaluated with samples collected at four points, two located in the natural channel and two in the rectified channel, during the four seasons of 2021. At each sampling point, with a van Veen dredge, three samples were collected for macrofauna and one for granulometric characterization. In total, 715 individuals were identified, and distributed in eight taxa. PERMANOVA revealed that the differences observed in the abundance of organisms considering seasonality were not significant, however, they were significant between the channels. The rectified channel exhibited 67% of the total abundance of the analyzed fauna. In this region, the organic matter and CaCO₃ exhibited the highest concentrations and the sediment was classified as silt, while in the natural channel, the granulometry varied between fine sand and silt. The oligochaete Tubificida was the most abundant organism, representing 97% of the fauna. Considered tolerant, this organism inhabits freshwater sediments with low oxygen concentration and high organic carbon load. The low richness of the community and the high dominance of Tubificida characterized the evaluated region as a highly altered environment.