Essential oils from the dried spikes of Nepeta tenuifolia (Benth) are obtained by steam distillation. Pulegone was identified as the main component in the spikes of N. tenuifolia through analysis, with greater than 85% purity obtained in this study. The essential oils are extremely active against all Gram-positive and some Gram-negative reference bacteria, particularly Salmonella enterica, Citrobacter freundii, and Escherichia coli. The minimum inhibitory concentration was found to be between 0.08 and 0.78% (against S. enterica), 0.39 and 0.78% (against C. freundii), and 0.097 and 0.39% (against E. coli), whereas the minimum bactericidal concentration varied in range from 0.097% to 1.04%. In general, the essential oils show a strong inhibitory action against all tested reference strains and clinical isolates. However, the antibacterial activity of EOs against both Pseudomonas aeruginosa reference strains and clinical isolates was relatively lower than other Gram-negative pathogens. The essential oils of N. tenuifolia also displayed bactericidal activities (MBC/MIC < 4) in this study. These findings reflect the bactericidal activity of the essential oils against a wide range of multidrug-resistant clinical pathogens in an in vitro study. In addition, we propose the fragmentation pathways of pulegone and its derivatives by LC-ESI-MS/MS in this study.