The Capibaribe River provides water to a population of 1.7 million people in the Brazilian northeast, while receiving agricultural runoff and industrial and domestic effluents along its 280 km. The present study evaluated the ecotoxicity of surface waters along ten sites in rural and urban areas using zebrafish (Danio rerio) early‐life stages and related it to water quality indices and chemical abiotic variables. Lethality rates, delays in embryo‐larval development quantified by the general morphology score (GMS), and frequencies of developmental abnormalities were analyzed. A correlation was detected between zebrafish GMS and water quality index (WQI), sensitivity to domestic sewage contamination, and trophic state index, focused on eutrophication. These indices agreed in identifying a spatial pattern of smaller impact in terms of ecotoxicity, domestic sewage contamination, and eutrophication risk at three sites in rural areas (mean GMS 16.9), an intermediate impact at four sites with urban and agricultural influence (mean GMS 16.4), and greatest impacts at three more urbanized sites (mean GMS 14.9). Most frequent developmental abnormalities included noninflation of the swim bladder, delayed hatching, nonprotrusion of the mouth, blood stasis, and nondevelopment of pectoral fins. Toxic NH3 concentrations varied spatially, with higher concentrations in urban sites; and blood stasis correlated positively with NH3, suggesting a causal relationship. Polycyclic aromatic hydrocarbons were detected in both rural and urbanized sites, contributing to detected toxicity. The present study demonstrates the potential of zebrafish early‐life stages as an ecotoxicological model that may contribute to a better understanding of surface water quality and ecotoxicity in tropical river systems. Environ Toxicol Chem 2021;40:3063–3077. © 2021 SETAC