Growing application of zinc oxide nanoparticles (ZnO-NPs) in global market has led to the concern over their potential environmental impacts. Filter feeders like mussels are prone to nanoparticles due to their superior filter-feeding ability. Temperature and salinity of coastal and estuarine seawaters often vary seasonally and spatially, and their changes may jointly influence physicochemical properties of ZnO-NPs and thus their toxicity. This study, therefore, aimed to investigate the interactive effect of temperatures (15, 25 and 30 °C) and salinities (12 and 32 PSU) on physicochemical properties and sublethal toxicity of ZnO-NPs towards a marine mussel Xenostrobus securis, and to compare that with the toxicity caused by Zn ions (zinc sulphate heptahydrate). The results revealed increased particle agglomeration but decreased zinc ion release of ZnO-NPs at the highest temperature and salinity condition (30 °C and 32 PSU). After exposure, ZnO-NPs significantly reduced survival, byssal attachment rate and filtration rate of the mussels at high temperature and salinity (30 °C and 32 PSU). Glutathione S-transferase and superoxide dismutase activities in the mussels were suppressed at 30 °C. These aligned with the augmented zinc accumulation with increasing temperature and salinity which could likely be attributable to increased particle agglomeration of ZnO-NP and enhanced intrinsic filtration rate of the mussels under these conditions. Together with the observed lower toxic potency of Zn compared to ZnO-NPs, our results suggested that the mussels might accumulate more zinc through particle filtration under higher temperature and salinity, eventually resulting in elevated toxicity of ZnO-NPs. Overall, this study demonstrated the necessity to consider the interactive effect of environmental factors such as temperature and salinity during the toxicity assessment of nanoparticles.