Resumo. AIM: This study aimed to determine the effectiveness of a Conservation Unit (CU) in maintaining the quality of freshwater habitats and to evaluate the influence of environmental integrity on benthic macroinvertebrate assemblages. METHODS: The research was conducted at sampling sites located within and outside of the CU in the Alto Uruguai region, southern Brazil, and included two stages: (i) the collection of benthic macroinvertebrates and (ii) the application of a Rapid Assessment Protocol (RAP) to characterise the habitat quality. RESULTS: A total of 1,362 benthic macroinvertebrates were collected during the study, totalling 30 taxa. The densities within and outside the CU were significantly different (F= 160.08; p= 0.05), and the Shannon diversity and taxa richness followed the same pattern (F= 118.72, p= 0.05; and F= 176.33, p= 0.04, respectively). In contrast, the Pielou equitability did not differ within and outside the CU (F= 0.19, p= 0.74). The biotic index (Biological Monitoring Working Party) indicated that water quality was good or very good in the majority of cases. Most of the sampling sites were classified as ‘natural’ according to the RAP. The taxa richness was significantly related to habitat diversity (F= 7.24; p = 0.05), but no significant relationship was found between the habitat diversity and the Shannon diversity (F= 2.13, p = 0.22). CONCLUSION: The CU was effective for the conservation of water quality and the freshwater biodiversity of benthic macroinvertebrates. The results show that the environmental integrity was related to the distribution of benthic macroinvertebrates, primarily to the taxa richness. More detailed investigations need to be developed to better understand these relationships and to take into account the temporal scale. An analysis of the most significant sources of stress on the aquatic life outside the area is recommended.