The sediment quality of Cádiz Bay, Las Palmas de Gran Canaria (LPGC) Port, Santander Bay, Algeciras Bay, and Huelva Estuary (Spain) was evaluated by analysing a battery of biochemical biomarkers-activities of biotranformation enzymes ethoxyresorufin O-deethylase [EROD], dibenzylflourescein dealkylase [DBF], and glutathione S-transferase [GST]; activity of antioxidant enzyme glutathione reductase [GR]; and lipid peroxidation [LPO]-in the polychaete Arenicola marina after laboratory sediment exposure. Huelva Estuary polychaetes showed significantly (p < 0.05) enhanced LPO, GST, and EROD activities compared with control lugworms related to metals and presumably polychlorinated biphenyls. EROD activity significant (p < 0.05) induction was associated polycyclic aromatic hydrocarbons after Santander Bay sediment exposure. Nickel appeared to significantly (p < 0.05) induce GR activity and LPO in LPGC Port sediment-exposed organisms. DBF activity significantly (p < 0.05) increased in polychaetes exposed to sediments from sewage-contaminated areas. A. marina was sensitive at the biochemical level. Integration of sediment characterization and biomarker results allowed the identification of polluted sites as well as the cause of possible sediment toxicity.