Over the last decade, the biological impact of microplastics in marine environments has become a serious global concern. Beyond their biological complexity, it is believed that many lethal and sublethal effects related to microplastic toxicity are triggered by oxidative stress and subsequently activated pathways. Thus, for marine organisms, having efficient mechanisms to fight the accumulation of oxidizing agents becomes indispensable to counteract the effects of microplastics. To date, our knowledge of the physiological effects of microplastics and the response of the antioxidant system in benthic species remains limited. The purpose of our study was to investigate the effects of short‐term exposure on the levels of two fundamental nonprotein antioxidants, glutathione (GSH) and ovothiol (OSH), in different tissues of Mytilus galloprovincialis. Our results show that the metabolism of OSH and GSH in mussels is clearly affected by acute microplastic exposure, and that the antioxidant response differs based on sex and reproductive stage. Indeed, although in the reproductive season the overall levels of GSH and OSH in different tissues significantly increase with respect to the control condition, the antioxidant response of the organisms, especially males, in the spent stage often exhibits a biphasic U‐shaped dose–response effect. Our study represents a pivotal investigation on the effects of microplastic exposure on the pools of two fundamental cellular antioxidants, with potential ecodiagnostic implications to forecast the stress status after exposure to microplastics, and highlights the possibility that the effects of these contaminants may vary over time based on the physiological condition of the animals. Environ Toxicol Chem 2023;42:1607–1613. © 2023 SETAC