AimsThe aim of this study was to screen a chemical library consisting of over 1200 pharmacologically active, already approved off-patent compounds, to determine whether any of the compounds reduced or eliminated the invasion or intracellular replication phenotypes of Salmonella enterica serovar Typhimurium (S. Typhimurium). Methods and ResultsLacZ reporter and tissue culture-based infection assays were used to screen for compounds that significantly reduced expression of key virulence genes, and were required for the invasion or intracellular replication phenotypes of S. Typhimurium in host cells. The search lead to the discovery of four compounds that reduced invasion by between 90-100%, and two compounds that reduced intracellular replication by between 65-93% at concentrations of either 2, 10 or 50gml(-1), relative to an untreated control strain. ConclusionsWe identified six compounds that significantly reduced expression of S. Typhimurium virulence genes resulting in decreased invitro virulence. Significance and Impact of the StudyThe emergence of multidrug-resistant strains of Salmonella poses a considerable and growing worldwide threat to human and animal health. The screening of off-patent chemical libraries represents a potential discovery route for novel antimicrobials.