Air velocity and temperature distributions inside operating rooms (ORs) play a crucial role to reduce the risk of infections and to ensure adequate comfort conditions for patient and medical staff. In this work, the authors have developed a three-dimensional thermo-fluid dynamic model to simulate airflow and thermal comfort in an actual OR equipped with High-Efficiency Particulate Air (HEPA) filters. The model takes into account the presence of surgical lights, people and equipment within the room. An experimental campaign is carried out inside the actual OR to measure velocity and temperature, to be employed as boundary conditions for the numerical model. The experimental data have also been used to validate the numerical results. The validated model has been used to analyze the effects of human shape, thermal boundary conditions and buoyancy forces on the main thermal and fluid dynamic quantities in the OR. The thermal comfort is evaluated based on Predicted Mean Vote (PMV) and Predicted Percentage of Dissatisfied (PPD) indices. The results prove that the present experimental-numerical approach is useful to analyze and improve the thermal comfort conditions for medical staff and patient.