The study of the toxicity, biocompatibility, and environmental sustainability of room-temperature ionic liquids (ILs) is still in its infancy. Understanding the impact of ILs on living organisms, especially from the aquatic ecosystem, is urgent, since large amounts of these substances are starting to be employed as solvents in industrial chemical processes, and on the other side, evidence of toxic effects of ILs on microorganisms and single cells have been observed. To date, the toxicity of ILs has been investigated by means of macroscopic assays aimed at characterizing the effective concentrations (like the EC50) that cause the death of a significant fraction of the population of microorganisms and cells. These studies allow us to identify the cell membrane as the first target of the IL interaction, whose effectiveness was correlated to the lipophilicity of the cation, i.e., to the length of the lateral alkyl chain. Our study aimed at investigating the molecular mechanisms underpinning the interaction of ILs with living cells. To this purpose, we carried out a combined topographic and mechanical analysis by atomic force microscopy of living breast metastatic cancer cells (MDA-MB-231) upon interaction with imidazolium-based ILs. We showed that ILs are able to induce modifications of the overall rigidity (effective Young's modulus) and morphology of the cells. Our results demonstrate that ILs act on the physical properties of the outer cell layer (the membrane linked to the actin cytoskeleton), already at concentrations below the EC50. These potentially toxic effects are stronger at higher IL concentrations, as well as with longer lateral chains in the cation.