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Abstract Background The flood in July 2021 is considered one of the largest flood disasters in Western Europe in decades, with massive socio-economic consequences. The potential emission and remobilization of anthropogenic pollutants can lead to additional environmental consequences, which need to be addressed in long-term mitigation strategies. The Inde River and its tributary, the Vichtbach River, form a catchment located at the transition from the low mountain ranges of the Eifel to the lowlands of the Lower Rhine Embayment in Germany. The area has been an industrial and mining hotspot for centuries, making it a high-risk area for flood sediment pollution. The present study provides an ecotoxicological screening of flood sediments of the Vicht–Inde catchment to gain an impression of the degree of contamination by organic pollutants by means of in vitro effect-based method. Sediment samples were collected within days after the flood and fractionated prior to biotesting, and supportive instrumental geochemical analysis was performed. Results Flood sediments did not reveal estrogenic potential, which was included in the testing strategy as a relevant endpoint for industrial chemicals and untreated wastewater. In contrast, moderate-to-high dioxin-like activity was observed in 70% of the sediment samples with a peak dioxin-like potential at the restored section of the Inde. Overall, four hotspot samples were identified as at risk, which aligned mostly with the high concentration of organic pollutants including PAHs and PCBs. The fractionation allowed the identification of PAHs and their derivates as the most likely toxicity drivers for dioxin-like activity in the sediments of the Vicht–Inde catchment. Conclusion The results provide first information on the prioritization of hotspot locations at risk for a detailed ecotoxicological profiling and a post-flood monitoring of organic contamination. The identified sinks of contamination in the floodplain areas can be considered a source for remobilization of pollution in future flood events, which is highly relevant for the receiving Rur River.