Quaternary ammonium salts (QASs) are irreplaceable membrane- active antimicrobial agents that have been widely used for almost a century. Cetylpyridinium chloride (CPC) is one of the most potent QASs, however, recent data from the literature indicate a 2- to 4-fold decrease in CPC activity against resistant bacterial strains. Given the growing demand for effective antimicrobials, especially in times of current and future spread of infectious diseases, the number of resistant isolates is expected to increase. One plausible approach to address this problem is to structurally modify the CPC structure by adding other biologically active functional groups. Here, a series of QASs based on pyridine-4-aldoxime were synthesized, characterized, and tested for antimicrobial activity in vitro. Although we obtained several potent antiviral candidates, Py- C12Br, Py-C12, and Py-C14, these candidates had lower antibacterial activity than commercial CPC. AFM images showed damage to the cell membrane and no viable cells after the bacteria were exposed to 4xMIC of Py-C12 for 3 hours. We found that the addition of an oxime group to the pyridine backbone resulted in an unfavorable electron density distribution and cLogP values and disrupted the interaction with the QacR dimer that regulates efflux pump expression. MD simulations showed that binding of Py-C16 to QacR leads to dissociation of the dimer within 50 ns, whereas the same was not observed in the case of the QacR dimer and the QacR dimer bound to CPC. This explains the lower bioactivity of our compounds, as they are likely to induce premature expression of the efflux pumps.