Pullorum disease, caused bySalmonellaPullorum (S. Pullorum), is one of the most serious infectious diseases in the poultry industry.Flos populiis traditionally used in Eastern Asian countries to treat various intestinal diseases. However, the anti-infection mechanism ofFlos populiis not very clear. In this study, we evaluated the anti-infective effects onS.Pullorum ofFlos populiaqueous extract (FPAE) in chickens. FPAE significantly reducedS.Pullorum growth in vitro. At the cellular level, FPAE reducedS.Pullorum adhesion and invasion on DF-1 cells but did not affect its intracellular survival or replication in macrophages. Further investigation revealed that FPAE inhibited the transcription of T3SS-1 genes, which is the main virulence factor that mediatesS.Pullorum adhesion and invasion in host cells. The results suggest that the anti-infective effect of FPAE likely occurs through the inhibition ofS.Pullorum T3SS-1, thereby impairing its ability to adhere to and invade cells. Further, we evaluated its therapeutic effect on animal models (Jianghan domestic chickens) and found that FPAE reduced the bacterial loads in organs and decreased the mortality and weight loss of infected chickens. Our findings provide novel insights into the potential development of FPAE againstS.Pullorum as an effective anti-virulence therapeutic substitute for antibiotics.