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Multimetric indices are considered a low-cost and rapid means of assessing ecological integrity in streams. This study aimed to develop a fish-based Index of Biotic Integrity (N3S-IBI) in an agricultural region within the domains of the Atlantic rainforest in Brazil. We sampled 23 first-order streams and used large-scale land use and a local physical condition index to choose reference sites and to classify sites according to the disturbance level. N3S-IBI resulted in six metrics (Simpson’s dominance; the numbers of Characiformes and non-native individuals (Poecilia reticulata); and the percentages of Characidae species, intolerant insectivorous individuals, and tolerant species), contemplating tolerance, composition, abundance, richness, trophic habits, and origin. The low number of metrics contributes to a quick and easy biomonitoring process. N3S-IBI showed an excellent performance to separate least and most disturbed sites in our study area and can provide additional knowledge about anthropogenic effects within this impacted region. In fact, this tool could be utilized by managers to direct restoration actions for the most disturbed sites and to strengthen the preservation of the least disturbed sites.