Climate change is predicted to result in rising average temperature of seawater with more extreme thermal events, and frequent rainfalls in some coastal regions. It is imperative to understand how naturally mediated changes in temperature and salinity can modulate toxicity of chemical contaminants to marine life. Thus, this study investigated combined effects of temperature and salinity on toxicity of zinc oxide nanoparticles (ZnO-NPs) to the marine diatom Thalassiosira pseudonana. Because ZnO-NPs formed larger aggregations and released less zinc ions (Zn2+) at greater temperature and salinity, toxicity of ZnO-NPs to T. pseudonana was less at 25 °C than at 10 °C and less at 32 than 12 PSU. However, toxicity of ZnO-NPs was significantly greater at 30 °C, since T. pseudonana was near its upper thermal limit. Three test compounds, ZnO, ZnO-NPs and ZnSO4, displayed different toxic potencies and resulted in different profiles of expression of genes in T. pseudonana. This indicated that ZnO-NPs caused toxicity via different pathways compared to ZnSO4. Mechanisms of toxic action of the three compounds were also dependent on temperature and salinity. These results provide insights into molecular mechanisms underlying the responses of the diatom to ZnO-NPs and Zn2+ under various regimes of temperature and salinity.