Effect-directed analysis (EDA) aims at identifying the compound(s) responsible for toxicity in a complex environmental sample where several dozens of contaminants can be present. In this study, we used an environmental mixture extracted from the Polar Organic Chemical Integrative Sampler (POCIS) previously immersed downstream a landfill (River Ponteils, South West France), to perform an EDA approach using a microalgal bioassay based on the photosynthetic capacities of diatom (Nitzschia palea) cultures. Adverse effects on photosynthetic capacities were recorded when algae were exposed to the entire POCIS extract (> 85% inhibition at the highest concentration tested). This result was coherent with the detection of diuron and isoproturon, which were the 2 most concentrated herbicides in the extract. However, the EDA process did not allow pointing out the specific compound(s) responsible for the observed toxicity but rather suggested that multiple compounds were involved in the overall toxicity and caused mixture effects.