The contamination of aquatic systems by endocrine disrupting chemicals (EDCs) is now a widely established fact. Nevertheless, there is still a scarcity of knowledge concerning the source, transport, fate and bioavailability of such active compounds. In the present study we assessed the distribution of estrogenic, (anti-)androgenic, pregnane X receptor-like (PXR) and dioxin-like activities between sediment and water compartments using a polar organic compound integrative sampler (POCIS) and a semi-permeable membrane device (SPMD) passive sampler in a river where sediment has been previously described as highly and multi-contaminated. We first confirmed the contamination pattern of this river sediment between 2004, 2009 and 2010 samples, suggesting that this river is subject to a constant high contamination level. However, we showed a different distribution pattern of these activities between compartments: estrogenic activity was mainly detected in POCIS extracts and to a lesser extent in sediment and SPMD extracts; anti-androgenic activities were mainly detected in SPMD and sediment extracts while no activity was detected in POCIS extracts; PXR-like activity was detected in all three investigated compartments, with POCIS > SPMD > sediment; dioxin-like activity was mainly found in the sediment and the SPMD extracts. Overall, partitioning of the biological activities was in accordance with physicochemical properties (e.g., log K ow) of typical known active chemicals in each bioassay. Furthermore, in order to establish whether the chemicals involved in these activities were similar between the compartments, we fractionated sediment, POCIS and SPMD extracts using a multi-step fractionation procedure. This highlighted differences in the nature of active chemicals between compartments. Altogether, our results support the need to consider different compartments in order to enhance exposure assessment.