Introduction: In mechanically ventilated patients, ventilator-associated pneumonia (VAP) is a major cause of prolonged hospitalization with increased morbidity and mortality. There is a lack of studies on the relationship between bacterial colonization of the ventilator circuit (VC) and VAP. This study aimed to investigate the role of bacterial colonization of VC in the development of VAP and identify antibiotic susceptibility trends for isolated strains. Methods: A prospective study of the bacterial culture has been performed between February 2021 to March 2021 on a total of 100 mechanically ventilated patients, (n =50) samples have been obtained from patient's lower respiratory tract (LRT) and (n =50) were taken from mechanical ventilator equipment VC. Paired samples of bacteria isolated from VC and LRT, where VC was colonized before LRT. Results: A total of 58 samples were cultured positively, while 42 specimens showed negative bacterial growth. However, there was no substantial difference in comparing between the bacterial colonization of the ventilator system and the patient samples. Most isolated organisms were gram-negative bacteria which were found in the ventilator circuit with 26 (68.4%), and 14 (70%) in patient’s LRT. Gram-positive was detected in 12 (31.6%) and 6 (30%) of the ventilator circuit, and patient's LRT, respectively. The predominant bacterial type was Acinetobacter baumannii organism at the VC with 10 (26.3%) and LRT at 4 (20%) followed by Klebsiella pneumoniae (8 (21.1%) in VC and 4 (20%) in LRT). Moreover, A. baumannii showed a full resistance to amoxicillin and the first generation of cephalosporins, while the other bacterial types were resistant to the most antibiotics used in this research. Conclusions: Bacterial colonization of ventilator circuit VC is a significant cause of VAP development in mechanically ventilated patients. Preventive strategies for the early detection and decontamination of contaminated VC can play a crucial role in ventilator-associated pneumonia prevention.