The main goal of ventilation in an operating room is to maintain air cleanliness in the surgical field and protect the patient from airborne contaminants. The most common ventilation systems used are the unidirectional system (which delivers air from the ceiling to the floor at low velocities) and the mixing ventilation system (which supplies air at higher velocities from ceiling or wall diffusers outside the surgical zone). Due to the different placement of air supply diffusers and the organization of flow patterns, different thermal conditions may be created in the field where the patient is located. The aim of the study was to describe the microenvironment conditions, depending on the ventilation system. However, unlike previous studies using thermal manikins, this research involved volunteers, both male and female. Research was conducted in a simulated operating room laboratory that supports both unidirectional and mixing ventilation. Fifty volunteers (25 female and 25 male) participated in the study. Measurements of air velocity were taken using five thermo-anemometers placed above the head, chest, abdomen, knees, and feet of the volunteers. The study was conducted under two different air temperatures (19 °C and 26 °C), with constant relative humidity (50%). The analysis of how different air supply conditions affect air velocity provided valuable insights that can help optimize operating room conditions. The conclusions aim to improve both the comfort and safety of patients and medical staff during surgeries.