As a primary cause of food contamination and human diseases, Salmonella Typhimurium can easily form a biofilm that is difficult to remove from food surfaces, and often causes significant invisible threats to food safety. Although berberine has been widely used as an anti-infective drug in traditional medicine, some basic principles underlying its mechanism, especially the interaction between berberine and type I fimbriae genes, has not been verified yet. In this study, two strains of major fimbrial gene mutants (Delta fimA and Delta fimH) were constructed to demonstrate the possible action of berberine on type I fimbriae genes. The broth microdilution method was used to determine the antibacterial activity of berberine against selected strains (WT, Delta fimA, and Delta fimH). Cell agglutination experiments revealed that the number of S. Typhimurium type I fimbriae reduced after berberine treatment, which was consistent with transmission electron microscopy results. Quantitative real-time PCR experiments also confirmed that berberine reduced fimA gene expression, indicating a certain interaction between berberine and fimA gene. Furthermore, confocal laser scanning microscopy imaging of biofilm clearly revealed that berberine prevents biofilm formation by reducing the number of type I fimbriae. Overall, it is well speculated for us that berberine could be an excellent combating-biofilm drug in clinical microbiology and food preservation.