Salmonella enterica serovar Typhimurium is an important foodborne pathogen that causes serious and extensive food contamination as well as disease and death worldwide. Considering the increasing severity of antibiotic resistance, antibiotic alternatives are urgently needed. As a natural biocide and a component of some essential oils from herbs, thymol is capable of killing various bacteria through a potentially unique mechanism, although the targets of thymol have not been completely elucidated. In this study, the variation in the whole proteome of Salmonella after thymol stress was evaluated using the SWATH multiplex technique. The strain Salmonella Typhimurium CVCC541 was treated with a sublethal concentration (75 mu g/mL) of thymol, which rapidly increased the permeability of bacterial membranes at the tested concentration. Thymol destroyed the integrity of the bacterial membrane, as observed by transmission electron microscopy. The proteomes of the treated and untreated cells were characterized after an 8-h treatment. The proteomic analysis of thymol-treated cells indicated that 144 proteins exhibited upregulation or downregulation compared with the control cells, particularly those involved in cellular structure and metabolism. The results of this study showed that thymol may play an antimicrobial role in altering the membrane permeability, virulence change, and antioxidant response of Salmonella Typhimurium. The results of the present study provide an improved understanding of the proteomic response of Salmonella Typhimurium to thymol stress, including the identification of promising targets for the future exploration of innovative approaches to control Salmonella Typhimurium.