Plastic debris has been a worldwide concern over the past years, wherein macro- and micro-plastics have been scientists' main focus. However, there still remains a scarcity of information on the effects that nanoplastics pursue on marine biota. This study aimed to comprehend the effects on 50 nm polystyrene nanoplastics (nPS, 10 µg/L) in an in vitro (24 h) and in vivo (21 days) exposure in the marine mussel Mytilus galloprovincialis. Characterization of nPS was conducted, whereby the ζ-potential and the hydrodynamic diameter were calculated in both freshwater and seawater. Mussel haemocytes were exposed in vitro to nPS and cell viability was assessed using the neutral red assay. In the in vivo exposure to nPS (10 µg/L), genotoxicity and oxidative stress were analysed in mussel gills. Results showed that nPS in freshwater had an average hydrodynamic diameter of 25 nm, with a ζ-potential of -68.4 mV, indicating that there is no aggregation. In seawater, on the other hand, the hydrodynamic diameter increases, indicating that aggregation does occur. ζ-potential of nPS in seawater is of -0.068 mV, meaning that in these conditions, as ζ-potential is closer to zero, aggregation is favoured. Results showed that turbidity is inversely proportional to sedimentation. Regarding the in vitro exposure, nPS decrease cell viability significantly (p