Pseudomonas aeruginosa is a notoriously difficult pathogen to control with antibiotics and disinfectants. Thymus vulgaris L. essential oil (TEO) and thymol are useful for replacing synthetic bacteriostatic agents due to their broad-spectrum bactericidal properties. This study focuses on understanding the inhibitory effects of TEO and its major component against P. aeruginosa, and exploring the antibacterial mechanism from the perspectives of cell membrane destruction, DNA intercalation and biofilm prevention. Results indicate that thymol is the principal component in TEO, as confirmed by GC-MS analysis. TEO and thymol potently combat the planktonic P. aeruginosa and directly change the cell structure. P. aeruginosa cell membrane integrity is destroyed as evidenced by an increase in permeability of the inner and outer membranes and the leakage of intracellular biological macromolecules (DNA). Our study further confirms that the accumulation of intracellular ROS caused by TEO and thymol can result in oxidative stress-mediated DNA damage. Thymol intercalates in the P. aeruginosa genomic DNA to affect the normal function of DNA and even cause bacterial death. Moreover, TEO and thymol affect the biofilm formation and limit the bacterial swimming and swarming motilities. This study provides a potential approach for the development and utilization of natural essential oils and bioactive compunds as novel food additives in the food processing.