Sha Yang,* Yun Yang,* Sixin Cui, Ziqi Feng, Yuzhi Du, Zhen Song, Yanan Tong, Liuyang Yang, Zelin Wang, Hao Zeng, Quanming Zou, Hongwu Sun National Engineering Research Center of Immunological Products & Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University of Chinese PLA, Chongqing, 400038, People’s Republic of China *These authors contributed equally to this work Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most predominant and fatal pathogens at wound infection sites. MRSA is difficult to treat because of its antibiotic resistance and ability to form biofilms at the wound site. Methods: In this study, a novel nanoscale liquid film-forming system (LFFS) loaded with benzalkonium bromide was produced based on polyvinyl alcohol and chitosan. Results: This LFFS showed a faster and more potent effect against MRSA252 than benzalkonium bromide aqueous solution both in vitro and in vivo. Additionally, the LFFS had a stronger ability to destroy biofilms (5 mg/mL) and inhibit their formation (1.33 µg/mL). The LFFS inflicted obvious damage to the structure and integrity of MRSA cell membranes and caused increases in the release of alkaline phosphate and lactate dehydrogenase in the relative electrical conductivity and in K+ and Mg2+ concentrations due to changes in the MRSA cell membrane permeability. Conclusion: The novel LFFS is promising as an effective system for disinfectant delivery and for application in the treatment of MRSA wound infections. Keywords: liquid film-forming system, methicillin-resistant Staphylococcus aureus, wound healing, antibiofilm, antibacterial