The aims of this study were to evaluate the effects of ajwain oil on multidrug-resistant (MDR) Enterobacteriaceae isolated from human faeces, the mode of action, and the active components. The studies also include the methicillin-resistant Staphylococcus aureus (MRSA). The ajwain oil showed broad spectrum and strong antibacterial activity against all the bacteria tested. The ajwain oil contained at least nine chemical compounds, and thymol was shown to be the most active component against all tested bacteria. Time-kill assay showed bactericidal effect and over 2 log reduction of MRSA and MDR Enterobacteriaceae after exposure to the ajwain oil for 30 and 10 minutes, respectively (1x, 2x, and 4x MIC). The scanning electron micrograph of MRSA and MDR Escherichia coli demonstrated bacterial cellular destruction after one-hour exposure to the 1x MIC of ajwain oil. Our results suggest that the application of ajwain oil may have potential for control and treatment of antibiotic resistant bacteria.