Background: Biofilm formation on endotracheal tubes (ETTs) is an early and frequent event in mechanically ventilated patients. The biofilm is anticipated to act as a reservoir for infecting microorganisms, contributing to the development and relapses of ventilator-associated pneumonia (VAP). Once a biofilm has formed on an ETT surface it is difficult to eradicate. The aims of this clinical study were to compare biofilm formation on three widely used ETT´s with different surface properties and to explore possible predictive factors of biofilm formation. Methods: The grade of biofilm formation on endotracheal tubes of polyvinyl chloride (PVC), silicone-coated PVC, and PVC coated by noble metals were compared after mechanical ventilation for > 24 hours in critically ill patients. The comparison was based on scanning electron microscopy (SEM) observations of ETT surfaces and biofilm grading, surveillance and biofilm cultures, and the occurrence of VAP. Results: A high-grade (score ≥ 7) biofilm formation on the ETTs was associated with the development of VAP (OR, 4.17; 95% CI 1.14 to 15.3; p = 0.031). Silicone-coated ETTs and PVC tubes coated with noble metals were independently associated with reduced high-grade biofilm formation compared to non-coated PVC ETTs (OR, 0.18; 95% CI 0.06 to 0.59; p = 0.005 and OR, 0.34; 95% CI 0.13 to 0.93; p = 0.036, respectively). No significant difference was detected between silicon-coated ETTs and noble metal-coated ETTs (OR, 0.54; 95% CI 0.17 to 1.65; p = 0.278). The microbes found in the ETT biofilm were frequently found in surveillance cultures at intubation and often remained in the biofilm after appropriate antibiotic therapy. Colonization with common VAP pathogens in surveillance cultures or duration of invasive ventilation did not predict higher biofilm formation on ETTs. Conclusion: Biofilm formation on ETTs were an early and frequent event in critically ill patients. High-grade biofilm formation on ETTs was associated with the development of VAP. Silicone-coated ETTs and PVC tubes coated with noble metals were independently associated with reduced high-grade biofilm formation in comparison to non-coated PVC ETTs. Clinicians may consider changing the ETT after an episode of VAP to avoid microbial persistence and possibly VAP relapse.