Aims: This study demonstrates the development of a mouthwash formulation containing the imidazolium salt (IMS) 1-n-hexadecyl-3-methylimidazolium chloride (C(16)MImCl), considering its stability and efficacy against Candida sp. Biofilm formation. Methods and Results: A variety of in vitro test methods were applied, assessing contaminated acrylic resin strip specimens before and after applying the mouthwash formulations. The formulation using C(16)MImCl presented a similar antibiofilm activity to cetylpyridinium chloride one and a commercial mouthwash, but at a 10 times lower concentration. Scanning electron microscopy imaging demonstrated that the selected mouthwash preparation fully destroys the biofilm cells, while with the hypoallergenicity test no irritant effect was observed in ex vivo model. Conclusions: The results presented herein indicate a high potential for imidazolium salts application as mouthwash agents that can eliminate Candida biofilm growth at very low concentrations. Significance and Impact of the Study: This study demonstrates a new and effective antibiofilm formulation containing the IMS C(16)MImCl. These findings suggest the IMS' use as mouthwash formulations active ingredient against Candida biofilms on oral surfaces, as it outperforms the often used cetylpyridinium chloride at a 10 times lower concentration.