Introduction: Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis in infants and one of the most common reasons for infant hospitalization, placing a substantial medical and economic burden globally . However, specific therapeutics for RSV infection remain limited. Objective: To identify safer and more effective agents for RSV prevention and treatment, and toelucidate their antiviral molecular mechanisms. Methods: We constructed recombinant RSV expressing mCherry fluorescent protein, screened D Usnic Acid via high throughput screening in HEp 2 cells, and validated its antiviral activity in vitro and in vivo using an RSV infection model. Small molecule– protein interactions were examined using biotin-avidin assay , surface plasmon resonance, and mass spectrometrybased cellular thermal shift assay . mRNA and protein levels of ABCB1, CTSS, TSPAN13 were detected by qPCR and Western blot. Result: D Usnic Acid showed significant anti RSV activity . It did not affect viral adsorption, entry , early replication, or translation, but inhibited RSV assembly and budding. WDR11 was identified as a potential key target, acting through the Hedgehog signaling pathway to regulate GLI family transcription. GLI1 modulated the expression of ABCB1, CTSS, and TSPAN13. Conclusion: D Usnic Acid exerts antiviral effects by targeting WDR11, which regulates the expression of ABCB1, CTSS, and TSPAN13 through the Hedgehog signaling pathway , thereby inhibiting RSV assembly and budding.