Benthic algae are the dominant primary producers in many rivers and may be at risk of being affected by sediment-associated toxicants. Toxicity as well as bioaccessibility should be considered for the identification of major toxicants affecting benthic algae. Thus, effect-directed analysis (EDA) combining biotesting, fractionation, and chemical analysis was combined with both exhaustive and bioaccessibility-directed extraction to identify the major toxicants in sediments at three industrial sites in the Elbe River basin (Bitterfeld, Most, and Prelouc). Exhaustive accelerated solvent extraction was used together with bioaccessibility-directed extraction with TENAX in aqueous suspension. Growth inhibition of the green alga Scenedesmus vacuolatus identified polycyclic aromatic compounds as important algal toxicants at all three sites, while in Bitterfeld and Prelouc additional toxicants, such as N-phenyl-2-naphthylamine and 7H-benzo[de]anthracen-7-one, played an important role. Confirmation of the identified compounds was based on the index of confirmation quality (ICQ) comparing the effect of an artificial mixture to that of the original sample. The results suggest a successful confirmation of identified toxicants for most fractions with ICQ values between 0.5 and 2. The bioaccessibility of toxicants decreased in the order Prelouc > Bitterfeld > Most as determined by the investigation of TENAX samples. Similar total concentrations of toxicants in Prelouc and Most sediment result in bioaccessible concentrations in Prelouc that exceed those in Most by a factor of 10 to 20. The extraction with TENAX with a subsequent EDA proved to be a powerful tool for prioritizing contaminants and contaminated sites with respect to the hazard of sediment-associated toxicants to green algae.