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Abstract Orthopedic foreign body-associated infection can be difficult to treat due to the formation of biofilms protecting microorganisms from both antimicrobials and the immune system. Exebacase is an antistaphylococcal lysin (cell wall hydrolase) under consideration for local treatment for biofilm-based infections caused by methicillin-resistant Staphylococcus aureus (MRSA). To determine the activity of exebacase, we formed MRSA biofilms on orthopedic Kirschner wires and exposed them to varying concentrations (0.098, 0.98, 9.8 mg/ml) of exebacase and/or daptomycin over 24 h. The biofilm consisted of 5.49 log10 colony forming units (cfu)/K-wire prior to treatment and remained steady throughout the experiment. Exebacase showed significant biofilm reduction at all timepoints (up to 5.78 log10 cfu/K-wire; P 3 log10 cfu/K-wire reduction) observed for up to 12 h for the 0.098 and 0.98 mg/ml concentrations and at 24 h for 9.8 mg/ml. Daptomycin showed significant biofilm reduction, although non-bactericidal, at all time points for 0.98 and 9.8 mg/ml and at 4 and 8 h with 0.098 mg/ml (P