Abstract: Salmonella typhimurium, a typical drug-resistant bacterium in livestock and poultry, has gradually developed resistance to the disinfectant benzalkonium chloride, causing a great impact on the agricultural and livestock industry. In order to slow down the resistance of Salmonella typhimurium to benzalkonium chloride, and explore the best conditions for killing Salmonella typhimurium with benzalkonium chloride, the method of re-suspension of bacteria with benzalkonium chloride of different media was used to screen the optimal medium for killing Salmonella typhimurium with benzalkonium chloride by the gradient dilution of spot plates. The results showed that the bactericidal effect of benzalkonium chloride would be largely inhibited if the anions produced by dissociation were present in the medium solution of benzalkonium chloride, which was also verified in Klebsiella pneumoniae and Staphylococcus aureus. Therefore, in the future production applications, the non-dissociated solutions could be used as the medium for benzalkonium chloride, thus to achieve the best bactericidal effect.