SUMMARY Diabetic foot ulcers (DFUs) are non-healing chronic wounds that develop upon skin injury in patients with poorly controlled diabetes mellitus (DM). One quarter of DM patients develop a DFU in their lifetime and, despite standard care, one in three DFUs fails to heal. DFU patients are highly susceptible to rapidly spreading infection that can lead to soft tissue damage and osteomyelitis, lower limb amputation (~100,000 annually in the USA), sepsis, and death. Most DFU infections are polymicrobial, and traditional therapies that target only one microorganism usually have a poor outcome in treating polymicrobial infections. Furthermore, polymicrobial DFU communities often form biofilms that are highly resistant to antibiotics. Currently, the standard DFU treatment is surgical debridement (i.e., removal of healing-impaired tissue harboring polymicrobial biofilms) and offloading to relieve the pressure from the affected foot. Despite treatment, DFUs often do not heal for weeks, months or even years. DFUs are treated with antibiotics when there are clinical signs of infection, but antibiotic therapy very often fails. Thus, DFU treatment is challenging and there is a need for new therapies. The goal of this application is to determine in vitro, ex vivo, and in vivo efficacy of the novel antimicrobial cyclic lipopeptide CLP4 on polymicrobial wound biofilms composed of the clinically relevant bacterial DFU isolates, and to determine the impact of CLP4 on the healing of infected wounds. In vivo local and systemic acute toxicity of CLP4 will also be determined. The results of this project will provide preclinical data for the development of an innovative DFU treatment.