Objectives: To investigate the level of bacterial contamination in ventilation devices after being connected to head trauma patients with confirmed ventilator-associated pneumonia. Methods: This prospective, cross sectional study was carried out at Shahid Mohammadi hospital, Bandar-e Abbas, Iran. Samples for assessing the contamination of ventilators were obtained from expiratory and inspiratory tube insertion sites before connecting the device to the patients. The patients were then observed for the development of ventilator-associated pneumonia and were enrolled in the study if considered eligible. Sampling was repeated after disconnecting the patient. The following variables were assessed in each patient: gender, age, Glasgow Coma Scale upon hospitalization, length of stay in the intensive care unit (ICU), and mortality. Results: A total of 33 patients, including 26 men and 7 women, were enrolled in the study. There was no significant association between ventilation contamination and time of sampling (before or after ventilation) in the evaluated sites (P > 0.05). However, based on McNemer’s test for equality of frequencies, the prevalence of positive culture after disconnecting the device from the patients (60.6%) was not the same as the prevalence before being connected to the patients (21.2%) at inspiratory tube insertion sites (P = 0.002). Also, at both sites, the variety and pathogenicity of microorganisms after disconnecting the device were higher than those of microorganisms, colonized from samples which were obtained before connecting the device. Conclusions: The findings of the present study showed that mechanical ventilation of patients with pulmonary infection leads to the contamination of ventilators. These findings suggest the need for designing and implementing new measures, which are easily available in developing and resource-deficient countries in order to reduce the contamination of ventilation devices and prevent cross-contamination.