The Estuarine-Lagoon Complex of Iguape-Cananeia (ELCIC), a Marine Protected Area (MPA) in Brazil, was the focus of this study that aimed to relate external levels of exposure to contaminants to toxic effects on Gobioides broussonnetii fish. Different anthropogenic contaminants such as metals, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs) were analyzed in the sediments; and biochemical, histopathological and genotoxicity biomarkers evaluated in fish; in two different seasons at three sites of the estuarine region. Higher contamination of the sediments was observed near the main urban center (Iguape city - IG). Metal concentrations were considered low to moderate, while PAHs concentrations were considered low. The concentrations of PPCPs increased due to the anthropogenic presence and were higher near IG and the Cananeia Island (CI). Contributions from historical mining, agriculture, nautical activities, oil, sewage and waste disposal, biomass and fossil fuels combustion were identified. Higher concentrations of metals and PPCPs were observed during the cold-dry season, suggesting influences of the lower hydrodynamics during the season of lower precipitation. Higher PAHs concentrations occurred in the hot-rainy season, indicating influences of greater human presence in summer. In fish, biological responses followed the same spatial and seasonal pattern. More pronounced changes in antioxidant, biotransformation, histopathological and genotoxic biomarkers were observed in IG and CI. The multivariate analysis and the integrated biomarkers response index (IBR) also evidenced worse environmental conditions in these sites. This result can indicate a negative influence of anthropogenic activities on the contamination of sediments and on the biological responses of fish. This study presented the first ecotoxicological data for the species and suggested that these chronic exposures can cause adverse effects on this fish population. The data contribute to the understanding of local environmental quality and can be applied in the future to the environmental and social management of marine protected areas.