Abstract This study presents a survey of small-stream fish species from the Purus-Madeira interfluve, collected in four streams near Humaitá on the highway BR-319. The results reveal a rich and diversified ichthyofauna with 3016 collected individuals distributed in 84 species, six orders, 25 families, and 60 genera. Of all the specimens collected, the Characiformes was the most representative, with eight families, 26 genera, and 42 species, followed Siluriformes, with nine families, 20 genera, and 23 species. In terms of families, Characidae had the highest number of species (25), followed by Loricariidae (9), and Cichlidae (8). Among the 95 captured species,s 11 are the first records for the region, evidencing a high diversity in these environments. Of the 84 species recorded in this study, 15 have not been assessed by the IUCN, while the remaining 62 include 23 listed as Least Concern (LC), three as data deficient (DD), and one as Near Threatened (NT). The southeastern Amazon region still has few fish surveys, especially in the region comprising the Purus-Madeira Interfluve, which highlights the importance of surveys to fill gaps and understand the biodiversity distribution patterns in the region.