In the context of supporting the EU Member States in the implementation of the EU Water Framework Directive (WFD), a project for the production of a fish reference material (ERM-CE100) certified for its content of the two priority substances hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) was carried out at the Joint Research Centre (JRC) of the European Commission. The starting material was naturally contaminated Wels catfish (Silurus glanis), caught in the Ebro River (Spain). A novel approach for the processing of the fish was tested that resulted in a homogeneous and stable reference material in the form of a wet paste. The fresh-like texture of the matrix enhances the comparability of this material toward routinely analyzed environmental biota samples and facilitates its use as a quality assurance tool given that the WFD environmental quality standards (EQS) for biota are expressed as wet weight. Certified values for the mass fractions of HCB and HCBD were assigned with 120 ± 8 and 36 ± 4 μg/kg, respectively. The related interlaboratory comparison involved 13 expert laboratories applying a range of analytical methodologies. It is the first biota CRM ever available for HCBD. ERM-CE100 can be used to assess the performance of analytical methods employed in the mandatory monitoring of water bodies under the WFD, thus, providing a benchmark for establishing comparability among measurement results.