Marine protected areas (MPAs) are vulnerable to many pressures, including pollution. However, environmental quality monitoring in these areas traditionally relies on only water chemistry and microbiological parameters. The goal of the current study was to investigate the role of a set of biomarkers in different target organs (liver, kidney, and gills) of fish in order to assess the environmental quality of an MPA (MTs, GPx, GST, GSH, DNA damage, LPO, AChE, and condition index). Chemical analyses were also performed on liver and muscle tissues to evaluate metal body burdens, and PAHs were identified in bile. A demersal fish (Cathorops spixii) that is widely consumed by the local population was used as bioindicator species, and the results were integrated using multivariate analysis. The use of the biomarker approach allowed for the identification of both seasonal and spatial variations in pollution sources around the Environmental Protected Area of Cananéia-Iguape-Peruíbe (APA-CIP). Higher metal body burdens associated with biological responses were found in the sites under the influence of urban areas during the dry season, and they were found in the sites under the influence of the Ribeira de Iguape River (RIR) during the rainy season. The liver was found to be more responsive in terms of its antioxidant responses, whereas gills were found to be more responsive to biomarkers of effect. These results show that this set of biomarker analyses in different organs of fish is a useful tool for assessing chemical pollution in an MPA.