The role of probiotics in the prevention of ventilator-associated pneumonia (VAP) remains inconclusive. The aim of this study was to assess the efficacy of a probiotic regimen for VAP prophylaxis in mechanically ventilated multi-trauma patients, intubated immediately after the injurious insult. In a randomized, placebo-controlled study enrolling multi-trauma patients, patients expected to require mechanical ventilation for >10 days were assigned at random to receive prophylaxis with a probiotic formula (n=59) or placebo (n=53). The probiotic formula was a preparation of Lactobacillus acidophilus LA-5 [1.75 × 10⁹ colony-forming units (cfu)], Lactobacillus plantarum (0.5 × 10⁹ cfu), Bifidobacterium lactis BB-12 (1.75 × 10⁹ cfu) and Saccharomyces boulardii (1.5 × 10⁹ cfu) in sachets. Each patient received two sachets twice daily for 15 days: one through the nasogastric tube and one spread on the oropharynx. The incidence of VAP was the primary endpoint. The incidence of other infections and sepsis, and the duration of hospital stay were the secondary endpoints. Administration of probiotics reduced the incidence of VAP [11.9% vs 28.3%, hazard ratio (HR) 0.34, 95% confidence interval (CI) 0.13-0.92; P=0.034] and sepsis [6.8% vs 24.5%, odds ratio 0.22, 95% CI 0.07-0.74: P=0.016]. Furthermore, probiotic prophylaxis reduced the time of stay in the intensive care unit (ICU) and the length of hospital stay. The prophylactic use of probiotics with a combination of enteral and topical application to the oropharynx had a positive effect on the incidence of VAP and sepsis, as well as on ICU and total hospital stay in patients receiving protracted mechanical ventilation.