To investigate the antibacterial effect and mechanism of flavonoids in Ampelopsis, network pharmacology was used to select key antibacterial flavonoid components in Ampelopsis and core targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to identify key signaling pathways, and molecular docking was performed to predict the interactions between key antibacterial flavonoids and core targets. The in vitro antibacterial activity of dihydromyricetin as a representative flavonoid was tested. Network pharmacology identified seven key components including dihydromyricetin, myricetin and kaempferol, five core targets such as prostaglandin-endoperoxide synthase 2 (PTGS2), tumor necrosis factor (TNF) and serine/threonine-protein kinase 1 (AKT1), and 23 key signaling pathways including the TNF signaling pathway and the C-type lectin receptor signaling pathway. Additionally, strong binding affinity between key components and core targets was observed. Dihydromyricetin exhibited a significant antibacterial effect on two strains of Salmonella, with minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 5 and 10 mg/mL, respectively. This flavonoid caused bacterial plasmolysis and alterations in the cell wall and membrane, and disrupted bacterial morphology. Therefore, flavonoids from Ampelopsis may regulate signaling pathways such as the TNF signaling pathway by acting on targets such as PTGS2 and disrupt bacterial morphology either directly or by influencing the bacterial environment, thereby exerting antibacterial effects.