BACKGROUND: Ventilator-associated pneumonia (VAP) is reported to occur in 12 to 25% of patients who require mechanical ventilation with a mortality rate of 24 to 71%. The endotracheal (ET) tube has long been recognized as a major factor in the development of VAP since biofilm harbored within the ET tube become dislodged during mechanical ventilation and have direct access to the lungs. The objective of this study was to demonstrate the safety and effectiveness of a non-invasive antimicrobial photodynamic therapy (aPDT) treatment method of eradicating antibiotic resistant biofilms from ET tubes in an in vitro model. METHODS: Antibiotic resistant polymicrobial biofilms of Pseudomonas aerugenosa and MRSA were grown in ET tubes and treated, under standard ventilator conditions, with a methylene blue (MB) photosensitizer and 664nm non-thermal activating light. Cultures of the lumen of the ET tube were obtained before and after light treatment to determine efficacy of biofilm reduction. RESULTS: The in vitro ET tube biofilm study demonstrated that aPDT reduced the ET tube polymicrobial biofilm by >99.9% (p<0.05%) after a single treatment. CONCLUSIONS: MB aPDT can effectively treat polymicrobial antibiotic resistant biofilms in an ET tube.