Ventilator-associated pneumonia (VAP) is a potentially preventable iatrogenic illness that may develop following mechanical ventilation. A bundle for the prevention of VAP consists of different measures which may vary between institutions, and may include: elevation of the head of the bed, oral care with chlorhexidine, subglottic suctioning, daily assessment for extubation and the need for proton-pump inhibitors, use of closed suction systems, and maintaining endotracheal cuff pressure at 25 cmH2O. Our aim was to determine the efficacy of a VAP prevention bundle, consisting of the above-mentioned measures, by evaluating the incidence of VAP before (no-VAP-B group) and after (VAP-B group) the introduction of the bundle. We retrospectively evaluated the data for patients who were mechanically ventilated with an endotracheal tube, in the period between 1 September and 31 December 2014 (no-VAP-B group, n = 55, 54.5% males, mean age 67.8 ± 14.5 years) and between 1 January to 30 April 2015 (VAP-B group, n = 74, 62.1% males, mean age 64.8 ± 13.7 years). There were no statistically significant differences between no-VAP-B and VAP-B groups in demographic data, intensive care unit (ICU) mortality, hospital mortality, duration of ICU treatment, and duration of mechanical ventilation. No significant differences in the rates of VAP and early VAP (onset ≤7 days after intubation) were found between no-VAP-B and VAP-B groups (41.8% versus 25.7%, p = 0.06 and 10.9% versus 12.2%, p > 0.99, respectively). However, a significant decrease in the late VAP (onset >8 days after intubation) was found in VAP-B group compared to no-VAP-B group (13.5% versus 30.9%, p = 0.027). Overall, our results support the use of VAP prevention bundle in clinical practice.