The estuarine ecosystem of Laguna Madre de Dios (LMD), in the Caribbean coast of Costa Rica, is exposed to contamination with pesticide residues coming from the upstream agricultural areas. Biomarkers can provide a better indication of the tness of biota in real mixture exposure scenarios than traditional lethal dose toxicity measurements. Here, we measured biomarkers of biotransformation, oxidative stress and neurotoxicity on Astyanax aeneus, an abundant sh species in LMD. Glutathione Stransferase activity (GST), catalase activity (CAT), lipid peroxidation (LPO), and cholinesterase activity (ChE) were measured in sh collected during seven sampling campaigns, carried out between 2016 and 2018. Pesticide residues were analysed in surface water samples collected every time sh were sampled.Residues of 25 pesticides, including fungicides, insecticides and herbicides, were detected. The biomarkers measured in A. aeneus varied along the sampling moments, however, biotransformation and oxidative stress signals showed coupled responses throughout the assessment. Furthermore, signi cant correlations were established between three biomarkers (GST, LPO and CAT) and individual pesticides, as well as between GST and LPO with groups of pesticides with shared biocide action. Among pesticides, insecticide residues had a major in uence on the responses observed in sh. This work shows that the frequent exposure to environmentally relevant concentrations of pesticides can be related to physiological responses in sh that affect their health. This early warning information should be considered to improve the protection of estuarine ecosystems in the tropics.