Air cleanliness is a crucial factor in operating theatres (OTs), where the health of patients and staff must be preserved by controlling air contamination. Particular attention must be paid to ultrafine particles (UFPs) size range, generated for instance by electrosurgical instruments (ESTs). OT contamination is also affected by ventilation systems, medical staff and their gowning system, staff routines, instruments, etc. This comparative study is based on experimental measurements of airborne microbial contamination and UFPs carried out during real ongoing surgeries in two OTs equipped with upward displacement ventilation (UWD) and hybrid ventilation, with unidirectional airflow on the operating table and peripheral mixing (UDAF+Mixing) ventilation systems. Airborne contamination concentration at the exit grilles has been analyzed as function of four different surgical phases normally performed during an operation. Results highlight that airborne contamination is influenced by the activities carried out during the surgical phases. EST usage affects the contamination level more than staff size during operation observed. Colony forming unit (CFU) values in the protected area close to the patient’s wound are influenced more by the type of ventilation system than by surgical phases. CFU values decrease by 18 to 50 times from the UWD system to the hybrid one. The large airflow volumes supply together with high air velocities in OTs equipped with UDAF+Mixing systems guarantee a better and a safer airborne contamination control for patients and medical team in comparison with UWD systems.