Environmental pollution by mercury (Hg) is of particular interest in Latin American countries like Ecuador, where artisanal and industrial gold mining has widely expanded. However, classic assessments, either based on water parameters or bioindicators, are not always effective in determining the effect of this type of pollutant on the ecological quality of the rivers. This study analyzed the behavioral and biochemical responses in benthic macroinvertebrate Nectopsyche sp. after sub-lethal Hg exposure emulating the maximum limits allowed by the current legislation in Ecuador (0.0002 mg L−1). Behavioral changes were significant, with an average decrease in the movement (measured as the number of strokes per 15 s of larvae) after 6 days of exposure to the maximum limit concentration of Hg. Furthermore, the antioxidant enzyme Glutathione S-Transferase activity significantly increased when exposed for 7 days to 0.0002 mg L−1 of Hg concentration. These results preliminarily proved that behavioral and biochemical patterns could function as biomarkers that efficiently identify ecological impairment caused by Hg, which would otherwise be neglected by a purely chemical-based or biomonitoring based on the presence/absence of macroinvertebrates type of assessment. Moreover, we demonstrate that the current limits allowed can impair aquatic life.