This study aimed to monitor an aquatic ecosystem during two different periods (dry and rainy season) on a protected area located inside a sugarcane farm, using the fish Astyanax sp. as bioindicator. An integrated approach was adopted by combining the responses of well-known biomarkers: acetylcholinesterase, lipid peroxidation (LPO), catalase (CAT), glutathione S-transferase (GST), micronucleus test, and liver histopathology. The activity of enzymes CAT and GST was increased after the rainy season. This can be explained mainly by the intensification of rain density, which drags substances into the streams, especially pesticides applied on agriculture. LPO and micronucleus test also suggested some effects of contamination in the surrounding area during this season. The results have supported a discussion about the effectiveness of protected areas in agricultural regions, emphasizing the biomonitoring as a tool for improving management plans in protected areas.