Copper is an essential metal for life. However, in excess, it can lead to osmoregulatory disorders and oxidative stress in fish and these effects appear to be species specific. In order to evaluate the effects of copper and to compare the sensitivity of two Neotropical fishes that co-occur in nature as prey (Astyaynax altiparanae) and predator (Hoplias malabaricus), the fish were exposed to three concentrations of Cu (5 μg L⁻¹, 10 μg L⁻¹, and 20 μg L⁻¹) for 96 h. At the end of the experimental period, copper concentration in tissues, osmoregulatory parameters, oxidative stress biomarkers, plasma glucose, muscle glycogen and acetylcholinesterase activity were evaluated. Fish mortality (25%) was only observed for A. altiparanae exposed to Cu 20 μg L⁻¹. The results revealed species-specific ionic disturbances. Despite hypocalcemia, H. malabaricus showed an increase in the main gill ATPases, which probably guaranteed the maintenance of plasma Na⁺. In A. altiparanae, there was no change in ATPase activity in the gills and hyponatremia was observed at all copper concentrations, as well as a decrease in plasma Cl⁻ in the Cu 20 μg L⁻¹ group. The strategy adopted by H. malabaricus seems to have contributed to the absence of copper accumulation in the tissues, in addition to possibly being related to the absence of oxidative stress in this species. On the other hand, there was an increase in the concentration of copper in the gills, liver, and gastrointestinal tract of A. altiparanae, as well as oxidative stress evidenced by increased lipoperoxidation in the liver and damage to erythrocytes DNA. This work reinforces the idea that copper effects are species specific and that a given concentration may not be safe for different species which can coexist in the same environment.