Background <p>Biofilm-associated lung infections, particularly those caused by <i>Staphylococcus aureus</i> (<i>S. aureus</i>), pose significant clinical challenges to conventional therapies. <i>S. aureus</i> Biofilm infections are refractory to treatment due to the presence of persister bacterial cells and the barrier effect of unique extracellular polymeric substances (EPS).</p> Results <p>This study describes the development of multifunctional micelles, HK-SL Ms, utilizing sophorolipid (SL) to encapsulate Honokiol (HK). HK-SL Ms potently disrupted the EPS barrier, killed some internal colonizing bacteria, and inhibited further bacterial adhesion. Consequently, the dynamic cycling of biofilms was hindered, achieving a promising removal of <i>S. aureus</i> biofilms. In vitro studies demonstrated that HK-SL Ms exhibited significant antimicrobial reduction of a 6.42 log₁₀CFU/mL. HK-SL Ms eradicated 71.73% of biofilms by targeting extracellular polysaccharides, extracellular proteins, and viable cells within the biofilm. Additionally, 1.66 log₁₀CFU/mL units of <i>S. aureus</i> within biofilms were killed. Moreover, HK-SL Ms inhibited 91.10% of early <i>S. aureus</i> biofilm formation by obstructing initial bacterial adhesion and the formation of extracellular polysaccharides and polysaccharide intercellular adhesins (PIA). Thus, the reestablishment and reinfection of <i>S. aureus</i> biofilms could be resolved promisingly. Biofilm infections are as predominant in acute pneumonia as in chronic cases, inducing similar lung inflammation. In a murine model of pneumonia infected by <i>S. aureus</i>, HK-SL Ms significantly reduced the bacterial load in the lungs, decreased inflammatory factor levels, and repaired lung tissue damage.</p> Conclusions <p>HK-SL Ms offers a novel strategy for the clinical treatment of biofilm-associated infections by dispersing and removing <i>S. aureus</i> biofilms and preventing new infections.</p> Graphical Abstract <p></p>