BACKGROUND Imidazolium salts (IS), ionic derivatives of neutral imidazoles, have properties that can be adjusted by structural modifications to their cations and anions, which makes this particular class of compounds a promising option for developing biologically active compounds. The anti-tumor effects of the IS 1-n-butyl-3-methylimidazolium chloride (C4 MImCl), 1-n-decyl-3-methylimidazolium chloride (C10 MImCl), 1-n-hexadecyl-3-methylimidazolium chloride (C16 MImCl), 1-n-hexadecyl-2,3-dimethylimidazolium chloride (C16 M2 ImCl), 1-n-octadecyl-3-methylimidazolium chloride (C18 MImCl), 1-n-hexadecyl-3-methylimidazolium methanesulfonate (C16 MImMeS), and 1-n-hexadecyl-2,3- dimethylimidazolium methanesulfonate (C16 M2 ImMeS) on oral squamous cell carcinoma (OSCC) have been studied here. METHODS Oral squamous cell carcinoma cells (CAL27) were incubated with increasing IS doses and then submitted to proliferation (2D), cell death (2D) and spheroid assay (3D). RESULTS The IS anti-tumor effect was dependent on both its N-alkyl chain length and anion, whereby C16 MImCl proved to be more effective in combination for inhibiting cell proliferation and cell-cell adhesion, outperforming the methylated C16 M2 ImCl derivative and, most importantly, the gold standard-cisplatin. In addition, C16 MImCl had little effect on keratinocytes and more pronounced effects on more aggressive tumor cells. It also exhibited similar effects on inducing cell death when compared to Cisplatin. This compound spread to a greater area of the tumor sphere and produced an enhanced number of apoptotic and necrotic cells in the tumor cell line, demonstrating only a small rise in the healthy cells. CONCLUSIONS These data indicate that the effect of C16 MlmCl on OSCC is promising, as it is selective for cancer cells.