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The susceptibility of streams to anthropogenic interference raises the need for continuous assessment of their environmental condition. From early studies to recent approaches, metrics derived from fish assemblages have proven to be fundamental tools in evaluating the ecological condition of watercourses. We assessed the environmental degradation of streams inside and surrounding the Córrego Grande Biological Reserve, state of Espírito Santo, Brazil, to develop and apply a biotic index for this region based on fish metrics. We performed samplings in 19 stream sites along 2012, 2018 and 2019, and collected 24 species belonging to five orders, 11 families, and 21 genera. The sites were classified as least-disturbed (N = 9, all within the protected area), intermediate (N = 8), and most-disturbed (N = 2). Ordination analyses distinctly separated the sites according to their disturbance classes, revealing that the percentage of native vegetation cover in 100 m buffers and type of riparian vegetation played an important role in the environmental quality of these sites. From the initial 38 metrics, three were able to distinguish between disturbance classes: percentage of Siluriformes and Characiformes species, percentage of nektonic species, and percentage of omnivorous individuals. Our results show that these metrics are significant factors to be considered in monitoring the environmental degradation of Atlantic Forest streams.