Mycoplasma synoviae (MS) is a wall-less pathogen primarily transmitted through the respiratory tract, causing synovitis and airsacculitis in poultry, which leads to substantial economic losses in the poultry industry. China has a long-standing tradition of Chinese medicine with abundant medicinal resources, renowned for its safety, efficacy, and holistic regulatory effects. This study aims to screen a traditional Chinese medicine (TCM) compound with anti-inflammatory and immunoregulatory properties and preliminarily validate its in vitro efficacy against MS infections. Based on the research foundation of this experiment and findings from previous studies, this study utilized network pharmacology analysis and molecular docking techniques to identify the anti-inflammatory and immunoregulatory effects of traditional Chinese medicinal herbs, including Rhizoma Coptidis, Honeysuckle (Flos Lonicerae Japonicae), Radix Codonopsis, and Radix Glycyrrhizae.The primary active components and potential targets were identified using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and other platforms. Protein-protein interaction (PPI) networks, Gene Ontology (GO) functional annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were employed to identify critical therapeutic targets. The AutoDock software was used for molecular docking between active components and core targets. Subsequently, in vitro experiments were conducted to validate the effects of key active components on MS targets. The analysis revealed that this TCM compound contains 122 active components, including flavonoids (e.g., quercetin, trimethylisoflavone), chromones (e.g., anti-inflammatory coumarin A), and triterpenes (e.g., glycyrrhizic acid B). These components act on 739 pharmacological targets, and Venn analysis identified 23 intersection targets related to MS. Further analysis indicated that STAT3, IL6, and IL1B were potential core targets. KEGG pathway enrichment analysis showed that MAPK, FoxO, and Toll-like receptor signaling pathways played critical roles in the pathology and treatment of MS, while GO analysis emphasized the significance of the NF-κB signaling pathway and VEGF receptor pathway in immune regulation, inflammation, and tissue repair. Molecular docking results demonstrated that lignans and quercetin in this TCM compound exhibited strong binding affinities to IL1B, IL6, and STAT3, potentially modulating these key targets to exert anti-inflammatory and immunoregulatory effects. In vitro experiments further validated the significant inhibitory effects of quercetin and glycyrrhizic acid on MS infection. The findings suggest that this TCM compound may exert therapeutic effects on MS infection by regulating targets such as CASP3, TLR4, STAT3, IL6, and IL1B, and modulating signaling pathways including MAPK, NF-κB, and FoxO.