The presence of pharmaceutically active compounds in aquatic environments has become a major concern over the past 20 years. Elucidation of their mode of action and effects in non-target organisms is thus now a major ecotoxicological challenge. Diclofenac (DCF) is among the pharmaceutical compounds of interest based on its inclusion in the European Union Water Framework Directive Watch List. In this study, our goal was to investigate the potential of a metabolomic approach to acquire information without any a priori hypothesis about diclofenac effects on marine mussels. For this purpose, mussel's profiles were generated by liquid chromatography combined with high resolution mass spectrometry. Two main metabolic pathways were found to be impacted by diclofenac exposure. The tyrosine metabolism was mostly down-modulated and the tryptophan metabolism was mostly up-modulated following exposure. To our knowledge, such DCF effects on mussels have never been described despite being of concern for these organisms: catecholamines and serotonin may be involved in osmoregulation, and in gamete release in mollusks. Our results suggest potential impairment of mussel osmoregulation and reproduction following a DCF exposure.