The analysis of functional diversity has shown to be more sensitive to the effects of natural and anthropogenic disturbances on the assemblages of aquatic macroinvertebrates than the classical analyses of structural ecology. However, this ecological analysis perspective has not been fully explored in tropical environments of America. Protected Natural Areas (PNAs) such as biosphere reserves can be a benchmark regarding structural and functional distribution patterns worldwide, so the characterization of the functional space of biological assemblages in these sites is necessary to promote biodiversity conservation efforts. Our work characterized the multidimensional functional space of the macroinvertebrate assemblages from an ecosystemic approach by main currents, involving a total of 15 study sites encompassing different impact and human influence scenarios, which were monitored in two contrasting seasons. We calculated functional diversity indices (dispersion, richness, divergence, evenness, specialization, and originality) from biological and ecological traits of the macroinvertebrate assemblages and related these indices to the physicochemical characteristics of water and four environmental indices (Water Quality Index, habitat quality, Normalized Difference Vegetation Index, and vegetation cover and land use). Our results show that the indices of functional richness, evenness, and functional specialization were sensitive to disturbance caused by salinization, concentration of nutrients and organic matter, and even to the occurrence of a forest fire in the reserve during one of the sampling seasons. These findings support the conclusion that the changes and relationships between the functional diversity indices and the physicochemical parameters and environmental indices considered were suitable for evaluating the ecological conditions within the reserve.