Summary Effects of various growth temperatures on cell membrane fatty acid composition of Salmonella Typhimurium (S. Typhimurium) and the resistance to Pulsed Electric Fields (PEF) treatments, as well as PEF combining with mild‐thermal (35, 45, 55 °C) treatments were investigated. Results indicated that the PEF resistance of S. Typhimurium at stationary phase was varied markedly at different growth temperatures. S. Typhimurium grown at 45 °C exhibited greater PEF resistance than cells grown at a lower temperature. Alteration of membrane fatty acid composition decreased in the unsaturated to saturated fatty acids ratio (UFA/SFA) and increased in cyclopropane fatty acids (CFA) proportion as growth temperature increased. It was found that the PEF resistance of S. Typhimurium at stationary phase was in a membrane fluidity dependent manner. Thermal combined PEF treatment improved the PEF lethality which indicated that the PEF resistance of S. Typhimurium was greatly affected by the fluidity of cytomembrane.