There is a multitude of pollutants that combine persistent and hydrophobic properties. In aquatic environments, they are largely deposited in sediments. The amount and characteristics of the organic carbon determine how strongly they are bound or if they are readily available for partitioning to biota and biouptake. These pollutants can be accumulated by aquatic organisms and biomagnified to higher trophic levels. Hence, it is important to explore the composition, activity and effects of environmental mixtures of pollutants in sediments of different origin, characteristics and pollution history. Sediments from Sweden, the European Arctic (coastal Svalbard vs. open sea), Queensland (Australia) and a French-German river were collected. The freely dissolved concentrations (Cfree) of the chemicals were determined using equilibration with thin coatings of silicone on the inner walls of glass jars with subsequent solvent extraction. Total sediment concentrations (Ctotal) were determined using accelerated solvent extraction. While there is a wide range of pollutants that have been detected in sediments world-wide, traditional chemical analysis cannot cover all compounds and their transformation products. Therefore, in this study, the extracts were dosed into seven cell-based bioassays covering cytotoxicity, activation of metabolic enzymes (binding to the arylhydrocarbon receptor, AhR), specific, receptor-mediated effects such as estrogenicity (ERa); and adaptive stress response (oxidative stress, AREc32). Cytotoxicity was assessed in all bioassays and occurred occasionally. Moreover, four of the seven bioassays were active in this study: AhR, AREc32, the peroxisome proliferator-activated receptor gamma (PPARg) and ERa. The activation of the AhR was by far most responsive and showed a distinct pattern across the sampling locations. The other three assays showed responses only at higher enrichment factors of the extracts, also revealing specific contamination patterns. A comparison between Cfree vs. Ctotal will enable assessing the actual risk (Cfree) vs. the potential hazard of those chemicals that might be released in future scenarios (Ctotal). The presented work calls for more detailed studies at specific sites and testing of additional endpoints with the aim of obtaining a complete picture of mixture effects caused by the freely dissolved and total concentrations of hydrophobic organic chemicals in sediments.