Extensive studies have shown that potassium diformate (KDF), an antibiotic substitute used as a feed additive, improves animal growth performance, although there is less direct evidence of its preventive effect on bacterial infections and its influence on the intestinal flora of animals. In this study, the inhibition effect of KDF on Salmonella enterica serovar Pullorum , an important enteric pathogen causing pullorum disease, was investigated in vitro and on a chicken infection model. The effect of KDF on the diversities and structures of chicken duodenal and cecum flora were also investigated using 16S rRNA gene sequencing. The results showed that addition of 0.5% KDF in feed or 0.1% KDF in drinking water significantly reduced the bacterial loads and the degree of pathological changes in the cecum, improved digestion and reduced the pH of the gastrointestinal tract of chickens infected with S. pullorum . KDF also significantly modified the diversity and abundance of intestinal microflorae in chickens. In particular, it promoted the colonization of several probiotics, such as Bacteroides , Blautia , Ruminococcus_torques_group and Faecalibacteriumm , which are involved in maintenance of the intestinal barrier, modulation of inflammation, energy supply for intestinal cells and pathogen resistance. These results enrich the theoretical basis for the clinical application of KDF in chickens.