In the present study microclimate and airflow patterns in a real operating theatre (OT) under effective use conditions are investigated. Surgical staff movements and sliding door opening/closing effects on the air thermal distribution and velocity fields are considered. Experimental measurements and numerical simulations are carried out for the “at rest” and “operational” conditions of the OT. Two “operational” use conditions are considered: “correct” and “incorrect” during a simulated hip surgery. Numerical and experimental results comparisons are used to check their compliance with the standard limits. Results mainly show the ventilation system effectiveness in providing the expected conditions, even though local perturbations due to operational conditions are clearly pointed out.