An accurate assessment of operative temperature inside a building is essential since it is associated to the human’s perception of well-being and comfort. In the present study, a 3D computational fluid dynamic (CFD) code is employed to evaluate the indoor operative temperature for a university office located in a historical building, consisting of thick masonry walls and large single glass windows, using fan coil as an air conditioning system. The experimental measurement was carried out using an innovative system for the sensor localisation (based on acoustic sources and a specific signal processing algorithm) in order to validate the numerical model and to obtain the required initial and boundary conditions for the case under study. The turbulent air flow, the operative temperature, and the local heat transfer characteristics inside the room are presented in typical winter conditions, allowing to identify the best comfort areas. The results show that the operative temperature of the room is significantly affected by surface temperatures of the windows, especially due to the large windows to wall surface ratio, and by the position and operational condition of fan coil. Comfort conditions inside the room are not uniform and big energy consumptions is required to maintain temperature set point. It is concluded that 3D comfort maps on operative temperature and air velocity allow to optimise internal layout of the office room; moreover the possibility to optimise comfort conditions in specific workstation together with control of operating mode of the fan coil (velocity and temperature of air inlet) allows to gain important energy saving results.