Abstract Astyanax is widely used in ecotoxicological bioassays and in biomonitoring studies because of the similarities that different species of the genus have regarding their ecological, morphological and behavioral characteristics. Due to these similarities, ecotoxicological studies are commonly conducted using any sampled Astyanax as if they were the same, i.e. without separating the species and using any gathering as a whole representative of the genus. This conduct is worrying since there are no studies showing the ecotoxicological relationship among the species. Therefore, this article addressed to this issue by inducing, through acute water exposure bioassays, the ecotoxicological responses of biomarkers of three species of Astyanax (A. altiparanae, A. bifasciatus and A. ribeirae). The antioxidant systems were induced by Cu contamination and were verified through the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and carbonic anhydrase (CA), while the responses of biotransformation (activity of etoxiresorufin-O-deetilase (EROD) and glutathione S-transferase (GST)), the neurotoxicity (activity of acetylcholinesterase (AChE)) and liver histopathology were checked against the contamination by malathion. Also, for both bioassays, the damage to macromolecules (lipids (LPO) and DNA (comet assay in blood (ECA) and liver (HCA), micronucleus test (MNA) and polichromatic erythrocytes (PCE)) were evaluated. Overall, the study showed that, despite the non-induction or the unclear action of some of the enzyme systems, all three species of Astyanax were susceptible to the contamination of both xenobiotics, since damages to their macromolecules were observed. From these results, a cluster analysis was performed and was efficient in integrating all the data of the three species to display the ecotoxicological relationship between them, which was consistent and representative. With this paper, we hope to contribute in promoting the investigation of the interspecifc relationship of ecotoxicological responses of Astyanax by creating a database on the genus and by encouraging further studies with this and other genera.