<p>Globally, aquatic ecosystems are facing the highest levels of anthropogenic pressures ever. Thus, the characterization of environments with little or no impact becomes critically important for the development of sound conservation efforts. The Santo Antônio River, a tributary of the Doce River, has already been proposed as a priority area for conservation as it is an important free-flowing river with high biodiversity in the Rio Doce basin. This study aimed to characterize the trophic structure of the fish assemblage in the Santo Antônio River using stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes. We evaluated the temporal stability of isotopic metrics using data from sampling conducted in 2020 and 2022. Over the 2&#xa0;years, a total of 24 fish species were collected. The fish community exhibited stability in isotopic parameters, despite temporal variation in species composition. The overall isotopic niche of the community varied minimally, showing substantial overlap (44%) between the 2-year values. The most important resources sustaining the community remained the same for most feeding guilds. Omnivores and invertivores primarily consumed aquatic invertebrates in both years, while piscivores relied on fish. Detritivores and herbivores fed mainly on periphyton in 2020 and on suspended material in 2022. Trophic diversity metrics remained stable. In general, despite some variations in collected species and assimilated items, the isotopic structure remained stable over time. Thus, these parameters can serve as a reference tool for comparison with other impacted sites in the basin and in so much-needed recovery efforts within the Doce River basin.</p>