Unidirectional flow air-conditioning systems are suitable for achieving indoor air quality required in high-performance operating rooms (organ transplants, cardiac surgery, aorta, orthopedic, burns, etc.). This paper analyzes indoor environmental conditions and technical and hygienic requirements for the design of heating, ventilation, and air-Conditioning (HVAC) systems for high-performance operating theatres. The main standards applicable to this type of operating rooms were compiled. Standard UNE 100713:2015, ASHRAE Standard 170-2017, and the European pre-norm EN 16244 were analyzed. A case study shows the lack of uniformity in design criteria and their ranges of values, which generates different design approaches. It was concluded that using these standards applicable to high-performance operating rooms leads to very different indoor environmental conditions for patients and medical personnel—air velocity profiles, air movement pattern, temperature, relative humidity gradients, and pressure stratification. Computational fluid dynamics (CFD) studies have been shown to detect areas of haven that favor microbiological load accumulation. In this way, it is possible to define cleaning protocols to attend these anomalies in a preventive way.