Background: Despite the clinical importance of ventilator-associated pneumonia (VAP) as the most common nosocomial infection in ICU, there are few studies in Iran evaluating the bacterial causative agents involving VAP. Objectives: The aim of the present study was to determine the prevalence of bacterial agents of VAP, and to evaluate the presence of S. pneumoniae in VAP- confirmed ICU patients by real-time PCR. Methods: In this cross-sectional study, during March 2016 to March 2017, 50 tracheal aspirates were collected from VAP-confirmed ICU admitted patients in Tehran. The number of epithelial cells and white blood cells (WBC) were determined by direct microscopy. Bacterial identification from VAP samples was done by routine biochemical tests and culturing on differential media. DNA was extracted from samples, and based on lytA gene amplification, a quantitative real-time PCR was performed for S. pneumoniae detection and quantification. Results: In culture, a pure bacterium was isolated from 40 out of 50 samples (80%), with Klebsiella pneumoniae (26%) and Acinetobacter ssp. (18%) being the most common isolates, respectively; however, all cultures were negative for S. pneumoniae. By real-time PCR, two samples (4%) were positive for S. pneumoniae with 4×104 and 1.6×105 CFU/mL bacterial load. These two samples contained 10 and 12 WBC/lpf, respectively. Conclusions: Although with only a with few clinical samples, this is the first study reporting pneumococcal VAP in Iran. Furthermore, in regards to the importance of VAP in ICU patients, more studies to optimize cultural method and evaluate applicable diagnostic molecular methods could be appreciated.