Ionic liquids (ILs) are composed of only anions and cations and are liquid solvents at room temperature. Different functional groups were introduced into the ILs, conferring them with specific functions or purposes and thus forming special ILs, namely task-specific ILs (TSILs). Imidazolium-based ILs are the most widely used ILs in industrial production. To date, there have been some studies on the toxic effects of ILs on different organisms. However, the effect of functionalized groups on the toxicity of ILs is still unclear. In the present study, zebrafish were used as model organisms to study the toxic effects of 1-ethyl-3-methylimidazolium nitrate ([C2mim]NO3) and 1-hydroxyethyl-3-methylimidazolium nitrate ([HOC2mim]NO3). The results showed that both promoted an increase in reactive oxygen species (ROS) contents, leading to lipid peroxidation and DNA damage. Furthermore, integrated biological response analysis showed that [HOC2mim]NO3 was less toxic to zebrafish than [C2mim]NO3, which indicated that adding functional groups decreased the toxicity of ILs to organisms. The influence of chemical structure on IL toxicity was also reported. These results could provide a scientific basis for better synthesis and utilization of ILs in the future.