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Bacterial biofilms, a critical chronic wound mediator, remain difficult to treat. Energy-based devices may potentially improve healing, but with no evidence of efficacy against biofilms. This study evaluates noncontact, low-frequency ultrasound (NLFU) in the treatment of biofilm-infected wounds. Six-millimeter dermal punch wounds in rabbit ears were inoculated with 107 colony-forming units ofPseudomonas aeruginosaor left as sterile controls. A biofilm was established in vivo using our published model. NLFU treatment was carried out every other day or every day, with contralateral ear wounds acting as internal, untreated controls. Wounds were harvested for several quantitative endpoints and scanning electron microscopy to evaluate the biofilm structure. TheP. aeruginosabiofilm consistently impaired wound epithelialization and granulation. NLFU, both every other day and every day, improved healing and reduced bacterial counts relative to untreated controls (p< 0.05). Scanning electron microscopy confirmed a qualitative decrease in bacteria after both treatments. NLFU also reduced inflammatory cytokine expression (p< 0.05). Our study suggests that NLFU is an effective therapy againstP. aeruginosawound biofilm. This represents the first in vivo evidence of energy-based modalities' impact on wound biofilm, setting the foundation for future mechanistic studies. Continued wound care technology research is essential to improving our understanding, and treatment, of biofilm-infected chronic wounds.