The Urdaibai estuary is a UNESCO biosphere reserve impacted by recreational, agricultural and industrial activities. Polycyclic aromatic hydrocarbons (PAHs) are major pollutants in Urdaibai, and their control and the identification of their sources is central on the preservation of the area. Chemical analysis by GC-MS showed a significant pollution by PAHs in Urdaibai sediment samples, mainly from pyrolytic sources, with minor contributions from oil spills. Measurement of the dioxin-like activity using a yeast-based bioassay showed an excess of biological activity in sediment samples from the inner part of the estuary. Analysis of PAH metabolites in bile of thicklip grey mullet (Chelon labrosus) showed a large excess of 2-naphthol over 1-naphthol or 1-pyrenol, suggesting a specific contamination in the inner Urdaibai estuary by some industrial process. Therefore, the combination of these three techniques defined different PAH pollution sources in Urdaibai: a major pyrolytic origin, occasional oil spills, and specific industrial activities.