Abstract <p>The growing antimicrobial resistance presents a challenge in developing new potent drugs, but this effort is hindered by a lack of information regarding how these new drugs would behave in biomembranes. Surfactants are considered mimetic models for biomembranes and can be used to study drug–membrane interactions. In this study, we used two well-known surfactants—cationic cetyltrimethylammonium bromide and anionic sodium dodecyl sulfate—as model membranes to investigate their interaction with the antimicrobial drug ofloxacin (OFL). These interactions were studied using volumetric and acoustic methods over the temperature range of 293.15–323.15&#xa0;K to determine the apparent molar volume, isentropic compressibility, apparent molar compressibility, acoustic impedance, relative association, and intermolecular free length. Furthermore, UV–Vis spectroscopy and cyclic voltammetry were employed to evaluate the binding constants and free energies of the drug–surfactant systems. These results provide key molecular insights into the thermodynamics of OFL partitioning and its binding mechanisms with amphiphilic assemblies. Such mechanistic understanding is crucial for the rational design of antibiotic delivery systems, facilitating precise control over drug loading and release dynamics in surfactant-based formulations.</p> Graphical Abstract <p></p>