As it is the case in all animal food production systems, it is often necessary to treat farmed fish for diseases and parasites. Quite frequently, fish farmers still rely on the aggressive use of copper to control bacterial infections and infestations by ecto-parasites, and to manage the spread of diseases. The susceptibility of the neotropical fish Rhamdia quelen to copper was here evaluated at different waterborne copper concentrations (2, 7 or 11μgCuL−1) for 96h, through a multi biomarkers approach. Liver histopathological findings revealed leukocyte infiltration, hepatocyte vacuolization and areas of necrosis, causing raised levels of lesions upon exposure to 7 and 11μgCuL−1. Decreased occurrence of free melano-macrophages and increased densities of melano-macrophage centers were noted upon exposure to 11μgCuL−1. Gills showed damages on their secondary lamellae already at 2μgCuL−1; hypertrophy and loss of the microridges of pavement cells at 7 and 11μgL−1, and increased in chloride cell (CC) apical surface area (4.9-fold) and in CC density (1.5-fold) at 11μgCuL−1. In the liver, catalase (CAT), glutathione peroxidase activities (GPx) and glutathione concentration (GSH) remained unchanged, compared to the control group. However, there was inhibition of 7-ethoxyresorufin-O-deethylase (EROD) at all copper concentrations tested. Glutathione reductase activity (GR) was reduced and levels of lipid peroxidation (LPO) were increased at 11μgCuL−1. Glutathione S-transferase activity (GST) at 7μgCuL−1 and superoxide dismutase activity (SOD) at both 7 and 11μgCuL−1 were reduced. However, copper exposure did not alter brain and muscle acetylcholinesterase (AChE) activity. Osmoregulatory function was also disturbed, in agreement with the above-mentioned changes noted in the gills, as detected by plasma osmolality reduction in the group exposed to 11μgCuL−1, and plasma chloride reduction at 2μgCuL−1. These concentrations also, coherently, lead to inhibition of branchial carbonic anhydrase activity. In the kidney, increased carbonic anhydrase activity was measured in the groups exposed to 2 and 7μgCuL−1. When these effects are compared to data available in the literature for other freshwater fish, also for 96h of exposure, R. quelen appears as a relatively sensitive species. In addition, the concentrations employed here were quite low in comparison to levels used for disease control in real culture practices (ranging from 4μgCuL−1 used against bacteria to 6000μgCuL−1 against fungal infections). We can conclude that the concentrations frequently employed in aquaculture are in fact not safe enough for this species. Such data are essential for the questioning and establishment of new policies to the sector. ► The susceptibility of the fish Rhamdia quelen to copper was evaluated/ R. quelen was shown to be a relatively sensitive species. ► Exposure to copper induces biochemical, morphological, and physiological effects. ► Concentrations frequently employed in aquaculture are in fact not safe enough.