<p>Fatty acids-based hydrophobic deep eutectic solvents (HDESs) have broad-spectrum antimicrobial activity. However, there is a lack of systematic studies on their antimicrobial mechanism at both experimental and molecular levels. In this work, we utilized hexanoic acid (HEA) and L-menthol (LST) to form HDES, and prepared HDES-in-water nanoemulsion, which is stable and effective in inhibiting the growth of <i>E. coli</i>. On this basis, we systematically investigated the antimicrobial mechanism of HDES against <i>E. coli</i>, examining the fluidity, integrity and permeability of cell membrane, morphological analysis of <i>E. coli</i> and molecular dynamics (MD) simulations. The results showed that HDES could decrease cell membrane fluidity and increase cell membrane permeability, which was further supported by MD simulations. In addition, HEA and LST can insert into the cell membrane, causing destabilization of the phospholipid bilayer, reduction of the membrane thickness, and disruption of the integrity of the cell membrane, leading to the efflux of intracellular material, which further promotes antimicrobial activity. The present work is important for the development of HDES-based natural antimicrobial agents to meet the increasing food safety challenges.</p> Graphical Abstract <p></p>