Although it was previously shown that prolonged prophylactic antibiotic exposure and multiple inadequate antibiotic therapies are independent risk factors for multidrug-resistant ventilator associated pneumonia there were no studies investigating whether pre-operative prophylactic dose of antibiotics changes oral microbiome and increases the risk of ventilator associated pneumonia. The aim of the study was to determine if pre-operative prophylactic dose of antibiotics affects the oral microbiome, increases the colonization with Gram-negative bacteria and subsequent risk of ventilator associated pneumonia. Mechanically ventilated adult patients receiving surgical antibiotic prophylaxis were included in the study. The presence of Gram negative microorganisms in the pre-prophylactic and post-prophylactic oral swabs and tracheal aspirates, as well as the occurrence of ventilator associated pneumonia, were analyzed. Number of patients colonized with Gram negative bacteria in post- prophylactic oral swab was significantly higher compared to oral swab taken before prophylactic antibiotic. On the other hand, the number of patients with Gram- negative bacteria in tracheal aspirates remained similar as in post- prophylactic oral swabs. Moreover, we found that presence of Gram- negative bacteria in both pre- and post- prophylactic oral swabs was in the positive correlation with the presence of Gram- negative bacteria in tracheal aspirates. This study showed increased colonization of oral cavity with Gram- negative bacteria after preoperative prophylactic antibiotics. Furthermore, receiving two prophylactic antibiotics from WHO Watch list increased the incidence of Gram- negative bacteria in oral swabs and tracheal aspirates, and the risk of ventilator associated pneumonia development.