In this study, a combination of network pharmacology, molecular docking, and experimental validation was used to jointly investigate the therapeutic mechanism of Bidens pilosa L. against bacterial diarrhea in chickens. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to identify the pharmacological components and targets of Bidens pilosa L., while the target genes related to bacterial diarrhea were analyzed in GeneCards, OMIM and DisgeNET databases, the intersecting targets were identified using the Venny database. Then, the 'Bidens pilosa L.-Component-Target-Diarrhea Network Diagram' was drawn by Cytoscape 3.10.2 software. STRING database was used to construct the protein-protein interaction (PPI) network and identify core genes; DAVID database was used to analyze GO enrichment and KEGG pathway enrichment; AutoDock was used for molecular docking validation; A chicken E. coli diarrhea model was established to observe the effect on intestinal tissue morphology and validate the target protein content using ELISA. The results showed that, the main active ingredients of E. coli for the treatment of chicken bacterial diarrhea were luteolin, coreopsin, 6, 7-dihydroxybenzofuran, and quercetin, corresponding to 215 target genes; After removing the duplicates, a total of 82 targets were intersected with the disease, and the main target proteins were interleukin-6 (IL-6), interleukin-1 beta (IL-1β), Caspase-3 (CASP3), Matrix metalloproteinase-9 (MMP9), and Hypoxia-inducible factor 1-alpha (HIF-1alpha); GO Functional enrichment analysis showed that the main targets of Bidens pilosa L.were related to apoptosis and inflammation; KEGG pathway enrichment analysis showed that the main targets of Bidens pilosa L. were concentrated in the signaling pathways such as HIF-1α, C-type lectin receptor, IL-17, TNF, and so on; Molecular docking results showed that Bidens pilosa L. had a good connectivity between the main active components and the key targets. Tissue section and ELISA results showed that Bidens pilosa L. significantly alleviated intestinal tissue damage and significantly reduced the levels of IL-6, IL-1β, CASP3, MMP9 and HIF-1α proteins in the jejunum (P < 0.05 or P < 0.000 1). The network pharmacology and experimental validation revealed that Bidens pilosa L. may regulate the development of intestinal inflammation by modulating the expression of IL-6, IL-1β, CASP3, MMP9 and HIF-1α, which in turn affects the signaling pathways such as HIF-1α, IL-17, TNF, etc., providing new ideas and methods for in-depth research on the mechanism of action of Bidens pilosa L. in the treatment of bacterial diarrhea in chickens.