This paper investigates the elimination of drugs of abuse from six different chemical classes and their metabolites in natural fluvial waters (nearby the output of a sewage system). Mineralization of these substances and toxicological characterization before and after treatment by a heterogeneous photo-Fenton system has been evaluated. This advanced oxidation technology was able to significantly reduce the concentration of the drugs of abuse in all the tested conditions (different hydrogen peroxide and catalyst loadings). However, toxicological analyses measured as inhibition of fern spore mitochondrial activity, showed only a complete elimination of acute and chronic toxicity when a higher solid catalyst loading was used (0.6 g/L). A lower catalyst loading of 0.2 g/L was not enough for toxicity elimination. These results evidence the need for combining toxicological tests and chemical analyses in order to establish the effectiveness of the water treatment technologies based on advanced oxidation processes.