Lidocaine hydrochloride (LH), a local anesthetic, and acetylsalicylate (AcSAL), show antibacterial activity for both gram-negative and gram-positive bacteria. Kinetic studies indicated that antibacterial activity of LH was different from that of AcSAL. A subinhibitory concentration of LH and AcSAL enhanced the sensitivity of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to novobiocin and nalidixic acid. The synergistic effect of AcSAL with novobiocin and nalidixic acid was higher than that of LH. The effect of both drugs on the membrane potential of inner membrane was also studied using inverted membrane vesicles of bacteria. Both LH and AcSAL depolarized the membrane potential after the vesicles were energized with nicotinamide adenine dinucleotide. However, unlike AcSAL, pre-treatment of vesicles with LH had no effect on the generation of membrane potential. These results suggest that depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for gram-negative bacteria, is associated with antibacterial activity of LH and AcSAL. The difference in actions of LH and AcSAL was discussed.