The Brazilian Pampa biome is currently under constant threat due to increase of agriculture and improper management of urban effluents. Studies with focus on the assessment of impacts caused by human activities in this biome are scarce. In the present study we measured stress-related biomarkers in tadpoles of the leaf frog Phyllomedusa iheringii, an endemic species to the Pampa biome, and tested its suitability as a bioindicator for the assessment of potential aquatic contamination in selected ponds (S1 and S2) nearby agricultural areas in comparison to a reference site. A significant decrease in acetylcholinesterase activity was observed in S2 when compared to S1 and reference. The levels of total-hydroperoxides were increased in S2 site. In parallel, increased activity of the antioxidant enzymes catalase, superoxide dismutase and glutathione S-transferase were observed in S2 when compared to S1 and reference. Further studies are necessary in order to correlate the changes observed here with different chemical stressors in water, as well as to elucidate mechanisms of toxicity induced by pesticides in amphibian species endemic to the Pampa biome. Nevertheless, our study validates Phyllomedusa iheringii as a valuable bioindicator in environmental studies.