Room-temperature ionic liquids (ILs) are considered green chemicals that may replace volatile organic solvents currently used by industry. However, toxicological effects of ILs are not well known. In this study, we describe the cytotoxicity of selected imidazolium-derived ILs in Caco-2 cells, prototypical human epithelial cells. The most toxic IL was 1-decyl-3-methylimidazolium chloride ([C₁₀mim][Cl]), whereas the least toxic was 1,3-dimethylimidazolium methyl sulfate ([C₁mim][MSO₄]). Using the toxicological experimental data obtained we developed a Quantitative Structure–Activity Relationship (QSAR) study using the Topological Sub-Structural Molecular Design (TOPS-MODE) approach. The model found showed excellent statistical parameters and from their interpretation, we arrived to some important conclusions such as: For 1-alkyl-3-methylimidazolium chloride derivatives, a correlation between the R₂ alkyl chain length and toxicity was observable. A positive contribution of p-chloro substituent in benzyl ring is detected among 1-methylarylimidazolium chloride derivatives. Regarding the contribution of the anion, anion chloride presents a positive contribution to toxicity whereas for compounds [C₁mim][MSO₄] and [C₁eim][ESO₄], a negative fragment contribution can be detected in anion methyl or ethyl sulfate. The EC₅₀ values determined for the ILs analysed in this study in Caco-2 cells are lower than the data reported in the literature on the toxicity of classical solvents assessed with cell lines. In conclusion, our results indicate that the possible cytotoxic effect of ILs should be considered in the design and overall evaluation of these compounds. Nevertheless, any toxicity evaluation of ILs should take their bioavailability into account, which will be affected by their low volatility, compared to conventional solvents.