Polycyclic aromatic hydrocarbons (PAHs) are contaminants often found in sediments. Their relevant contribution to toxic effects induced by environmental samples has been demonstrated using effect-directed analysis (EDA). Toxicity of PAHs previously has been reported to depend on light conditions. The aim of this study was to analyze the influence of simulated sunlight, in comparison to standard algal growth light, on the toxicity of samples, in which PAHs were identified as major toxicants using EDA. Additionally, toxicity of identified toxicants and mixtures of these compounds were assessed. It can be shown that the samples, the PAH compounds, and the created mixtures exhibit photoenhanced toxicity. The combined effects of the mixtures can be predicted using the model of concentration addition. This is surprising because different modes of action have been reported to contribute to phototoxicity of the identified toxicants. For the confirmation step in EDA, the toxicity assessment under simulated sunlight reveals that less of the samples' toxicity can be explained by the identified compounds, compared to the assessment conducted under standard growth light. Finally, the relevance of light conditions in the toxicity assessment is demonstrated for eight out of 13 transect samples of sediment extracts from river Elbe, Germany.