Offshore petroleum exploration has increased the risks of oil spills in coastal tropical and subtropical habitats. Monitoring tools are needed to assess and protect environmental health. We determined baseline values of antioxidant biomarkers (CAT, SOD, GPx, GST, MDA) for five ecologically relevant species in a subtropical system in southern Brazil. Regional baseline levels are compared with literature data as a basis to eventually test their efficacy as post-spill monitoring tools. Differences in the antioxidant response among species, contamination, and seasons were tested using univariate and multivariate analyses. The bivalves Anomalocardia flexuosa and Crassostrea rhizophorae and the catfish Genidens genidens emerge as suitable sentinel species. Seasonality is the main factor accounting for biomarkers variability, and not background contamination level. However, interactions between season and contamination level are also significant, indicating that biomarkers respond to complex environmental settings, a fact that needs to be fully understood for designing proper monitoring programs.