Reactive oxygen species present a challenge for marine organisms releasing gametes into the water. Thiol-containing molecules protect cells against oxidative stress, and ovothiol (OSH), an antioxidant-reducing mercaptohistidine, has been described as especially relevant in the oocytes of marine invertebrates. Ovothiol synthase (ovoA), in charge of the first step in OSH synthesis, was sequenced in mussels, Mytilus galloprovincialis. Transcription levels of ovoA in mantle did not significantly change along the reproductive cycle. No alterations of ovoA transcription were observed after a laboratory copper (10 µg/L) exposure or in mussels captured in a highly polluted site. Conversely, the metabolomic analysis of the hydrophilic metabolite content in mantle clearly classified mussels according to their site of origin, especially at the most advanced stages of oogenesis. Quantification of OSH-A and -B and glutathione (GSH), revealed stable levels in mantle at early gametogenesis in the unpolluted sampling site, but a strong increase in female mantle previous to spawning in the polluted site. These increased concentrations under pollution suggest that OSH-A accumulates along oogenesis, independent of gene transcription regulation. The concerted accumulation of OSH-A and GSH suggests the building of a balanced cellular redox-system to scavenge ROS produced in the oocyte before and during fertilization.