The clam Ruditapes decussatus is an important resource to preserve in coastal lagoon systems around the Mediterranean including the South Portugal. To assess spatial and temporal biomarker responses to contamination in the species, a multibiomarker approach was conducted using antioxidant enzymes, MFO system phase I and II; acetylcholinesterase, metallothionein (MT), δ-aminolevulinic acid dehydratase and lipid peroxidation (LPO). The condition index (CI), metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. The levels of contaminants were not particularly high and the antioxidant enzymes, acetylcholinesterase (AChE), MT in the digestive gland, and δ-aminolevulinic acid dehydratase (ALAD) do not provide a suitable seasonal and spatial discrimination reversely to that regarding CYP450, glutathione-S-transferase (GST), MT in the gills, and LPO in both tissues. However, even those could vary with natural variables that may act as confounding factors. Thus, seasonal variability and natural range of biomarker responses must be carefully and accurately taken into account in ecotoxicological approaches of environmental quality assessment programmes.