With more than 7000 inner rivers, streams, small permanent or temporal watercourses, and wetlands, Entre Ríos province has the richest fish diversity in Argentina due to habitats diversity generated by the climatic, biogeographic, and topologic characteristics. However, this region is one of the most impacted areas by intense livestock and farming activities. For this reason, it is necessary and urgent to assess the fish richness and biotic integrity to strategically conserve resources. The aims of this study were to study the fish assemblages of a temperate third order stream namely Santa Barbara, and to determine the environmental quality through modified Index of Biotic Integrity (IBI). The study area is located in the lower-middle basin of the stream in the south-east of Entre Ríos (Gualeguaychú department). Fish samples were collected and identified in 3 sampling stations during 2012 (n = 5), and 11 metrics were calculated. One hundred and sixty-seven native organisms were collected, and 17 species, 3 genera, and 1 family were identified. Presence of Serrapinus microdon was reported, representing the most austral record in Argentina and the first record in Entre Ríos province. Modified IBI was applied to fish data and showed that the study area exhibited between poor and good biotic integrity. This work contributed to the knowledge of ichthyofauna from inner sub-basin of the Uruguay river and it was the first application of IBI in the Mesopotamic Pampa with an adaptation proposal.