Mechanical ventilation using endotracheal tube (ETT) intubation is crucial in saving life but may also cause ventilator-associated pneumonia resulting in morbidity and mortality. The purpose of this study was to examine the effects of intubation duration on pathogen colonization rates of ETT cuff region, and its association with the subsequent re-intubation and tracheostomy. We enrolled 92 patients who were successfully weaned from ventilator and were extubated within 20 days of intubation duration. Patients were divided into Group I and II based on intubation for 1-9 days and 10-20 days, respectively. Pathogen colonization over ETT cuff region and extra-cuff region (including sputum and ETT aspirates) were assessed. As compared to Group I patients, Group II patients had a significant higher pathogen colonization rate (100% vs. 69.2%; P < 0.001) in the ETT cuff samples, but not in the extra-cuff samples (92.6% vs. 84.8%; P = 0.442). Further studies demonstrated that there was no difference between Group I and II patients in the percentages of patients with the same pathogen over both the cuff and extra-cuff samples (35.5% vs. 30.8%; P = 0.925), suggesting that the increased pathogen colonization rate over the ETT cuff region was least likely from the extra-cuff region. In addition, the results showed that longer intubation was also associated with increased tracheostomy rate from 9.3% to 28.9% for Group I and Group II respectively (P = 0.025). We conclude that longer intubation has a higher pathogen colonization rate over the ETT cuff region in patients receiving mechanical ventilation support; longer intubation also increases the trend of receiving re-intubation and tracheostomy. Our findings indicate that it is crucial to remove ETT as soon as possible and perform pathogen culture over the ETT cuff regions immediately after extubation.