Background: Ventilator-associated pneumonia (VAP) is the commonest infection in critically ill patients. The definite mortality rate of VAP is still controversial but may exceed 50% when the initial treatment is not appropriate. Objective: was to compare the performance of quantitative real-time PCR with semi-quantitative culture for diagnosis of VAP caused by P. aeruginosa using two sample types bronchoalveolar lavage and endotracheal aspirates (BAL and ETA) and to phenotypically determine some of P. aeruginosa virulence factors associated. Methodology: Two samples were collected from every patient on the day of suspected VAP, one is endotracheal aspirate sample and the other is bronchoalveolar lavage sample. Each sample (BAL and ETA) was divided into two aliquots, one for conventional microbiological analysis and the other was frozen at −80°C for molecular detection and quantification of P. aeruginosa. Isolates were phenotypically tested for the expression of some of the virulence factors frequently associated with human infections. Results: There was excellent concordance correlation between qPCR and conventional culture for detection of P. aeruginosa in BAL samples, between both methods for detection of P. aeruginosa in ETA samples and between the two types of samples (BAL and ETA) for the two investigated methods. The most frequently associated virulence factor was alkaline protease production in 81.25% of isolates, followed by biofilm forming capacity in 75% of the isolates. Conclusion: Our study revealed that qPCR can afford rapid and reliable quantitative microbiological data, with very high sensitivity and specificity for P. aeruginosa involved in VAP. It also tended to verify that the same pathogen can be detected and quantified from the two sample types using conventional or molecular method. These results decrease the utility of BAL sample, which is difficult to perform, more invasive, expensive and more time consuming than ETA.