Purpose Ventilator-associated pneumonia (VAP) is one of the most common infections in intensive care units (ICU) with a 6–52% incidence. The VAP mortality rate is 50% to 70%. Medical professionals (MPs) working in the ICU are expected to follow the guidelines to prevent VAP. The study aimed to assess the performance of MPs in preventing VAP and to associate the performance with the baseline information. Methods An observational cross-sectional study was conducted in the ICUs of selected hospitals in eastern Saudi Arabia. A total of 152 MPs were selected by random sampling. A structured questionnaire including baseline information, knowledge and performance-related questions was used to collect the data. Frequency, mean, and chi-square tests were used for analysis. Results Out of 152 MPs, 40.8% had adequate and 7.9% had inadequate knowledge. A high mean score of 12.9 ± 2.2 was obtained by physicians, followed by 11.3 ± 1.6 by nurses, 9.8 ± 2.2 by RTs, and 8.6 ± 2.1 by interns. Overall, 52.6% had satisfactory performance. Approximately 57.9% and 67.8% of MPs cleaned their hands before touching the patient and the ventilator, respectively. Many (79.6%) MPs used personal protective equipment in the ICU. Some (47.4%) of the MPs changed the patient’s position regularly. About 77.6% of MPs followed the sterile technique when suctioning the airway. There was a significant association found between the performance of MPs on the prevention of VAP with age ( p < 0.001), designation ( p < 0.05), professional experience ( p < 0.05), managing chronic obstructive pulmonary disease conditions ( p < 0.05) and training attended ( p < 0.001). Conclusion Although some of the MPs had satisfactory performance regarding VAP prevention in the ICU, more attention should be paid to training them on clinical guidelines to improve health care quality and reduce the rate of VAP.