OBJECTIVE: To evaluate the effects of systemic treatment with linezolid compared with vancomycin on biofilm formation in mechanically ventilated pigs with severe methicillin-resistant Staphylococcus aureus-induced pneumonia. DESIGN: Prospective randomized animal study. SETTING: Departments of Pneumology, Microbiology, and Pharmacy of the Hospital Clínic, Barcelona, and Scientific and Technological Services of the University of Barcelona. SUBJECTS: We prospectively analyzed 70 endotracheal tube samples. Endotracheal tubes were obtained from pigs either untreated (controls, n = 20), or treated with vancomycin (n = 32) or linezolid (n = 18). INTERVENTIONS: The endotracheal tubes were obtained from a previous randomized study in tracheally intubated pigs with methicillin-resistant Staphylococcus aureus severe pneumonia, and mechanically ventilated for 69 ± 16 hrs. MEASUREMENTS AND MAIN RESULTS: Distal and medial hemisections of the endotracheal tube were assessed to quantify methicillin-resistant Staphylococcus aureus burden, antibiotic biofilm concentration by high-performance liquid chromatography or bioassay, and biofilm thickness through scanning electron microscopy. We found a trend toward a significant variation in biofilm methicillin-resistant Staphylococcus aureus burden (log colony-forming unit/mL) among groups (p = .057), and the lowest bacterial burden was found in endotracheal tubes treated with linezolid (1.98 ± 1.68) in comparison with untreated endotracheal tubes (3.72 ± 2.20, p = .045) or those treated with vancomycin (2.97 ± 2.43, p = .286). Biofilm linezolid concentration was 19-fold above the linezolid minimum inhibitory concentration, whereas biofilm vancomycin concentration (1.60 ± 0.91 μg/mL) was consistently below or close to the vancomycin minimum inhibitory concentration. Biofilm was thicker in the vancomycin group (p = .077). CONCLUSIONS: Systemic treatment with linezolid limits endotracheal tube biofilm development and methicillin-resistant Staphylococcus aureus burden. The potential clinical usefulness of linezolid in decreasing the risk of biofilm-related respiratory infections during prolonged tracheal intubation requires further investigation. Copyright © 2012 by the Society of Critical Care.