This paper presents the adaptation of Escherichia coli Gram-negative bacteria to increased concentrations of ionic liquids. Theophylline-based quaternary ammonium salts were used as an example of an ionic liquid that on the one hand includes an anion of natural origin and on the other hand is characterized by amphiphilic properties due to aliphatic chains in its structure. Theophylline-based ionic liquids can be synthesized relatively cheaply and easily and can exhibit strong antibacterial properties depending on the alkyl chain length. These compounds can also strongly affect bacterial membrane properties, including changes in electrokinetic potential as well as net surface charge. The experiments performed in this study succeeded in obtaining bacterial cultures growing at a tetradecyltrimethylammonium theophyllinate concentration three times higher than the minimum inhibition and bactericidal concentration. The adapted bacteria were characterized by intriguing changes in morphology and grew in the form of almost one-millimeter spheres in a liquid medium. It was shown that cultivation of adapted bacteria with tetradecyltrimethylammonium theophyllinate resulted in changes in the lipid membrane composition and protein patterns of the bacterial lysates, depending on the ionic liquid concentration. This study also revealed that such bacterial adaptation can increase sensitivity to antibiotics by affecting membrane properties like ionophores. These results can be potentially important with regard to synergistic or antagonistic action with other bactericidal compounds like antibiotics and nanoparticles.