Abstract
Background/Objectives: Deep eutectic solvents (DESs) have recently gained attention for their antimicrobial properties, particularly because they target both planktonic bacteria and biofilms. Among these, DESs based on α-hydroxy acids (αHAs) are of interest due to their inherent antibacterial properties and favorable biocompatibility. However, effects of the αHA molecular structure and hydrogen bonding ability within a DES formulation on biological activity has not yet been thoroughly investigated. Methods: This study systematically investigates DESs formed by combining glycolic acid, lactic acid or tartaric acid with either choline chloride or tetraethylammonium chloride. Results: All DESs demonstrate broad-spectrum antibacterial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa and effectively inhibit biofilm formation while exhibiting low cytotoxicity toward 3T3-L1 fibroblasts. Conclusions: DES formation enhances antibacterial efficacy while attenuating cytotoxicity compared to the individual components, thereby decoupling bactericidal activity from toxicity. Physicochemical characterization confirms the formation of a eutectic phase and reveals that biological activity is primarily governed by acidity rather than by the specific αHA structure or eutectic strength. These results provide new insights into structure-function relationships in DESs and establish a design strategy for biocompatible, non-cytotoxic antimicrobial agents.