The Atlantic Rainforest, a biodiversity hotspot, has undergone significant fragmentation, with only 11% to 16% of its original cover remaining. Freshwater ecosystems within this rainforest are essential habitats, hosting high levels of biodiversity. This study assessed water quality and freshwater invertebrate diversity in rivers within and outside protected areas of the Non-Governmental Organisation Iracambi in Minas Gerais, Brazil, during the wet season from September to December 2022. We hypothesised that rivers with riparian vegetation would exhibit higher biotic indices and overall better water quality compared to unprotected rivers. Abiotic parameters air and water temperature, salinity, pH, conductivity, dissolved oxygen, flow rate, and nitrate concentration, were measured across 16 river sites, and freshwater invertebrates were surveyed to calculate Biotic Indices based on pollution tolerance. Results indicated that protected sites had a significantly higher mean biotic index compared to unprotected sites, suggesting better water quality in protected areas. Species richness was also higher in protected rivers, with 16 taxa identified compared to 11 in unprotected rivers. Pollution-tolerant species, such as Annelida and Argulidae, were more prevalent in unprotected rivers, while pollutant-intolerant species, such as Plecoptera and Spinadis, were more common in protected rivers. These findings highlight the importance of riparian vegetation in maintaining water quality and biodiversity, highlighting the need for ongoing monitoring and conservation initiatives in the Atlantic Rainforest especially in remote or isolated areas.