Reducing energy consumption in buildings has received intensified research impetus since the introduction of the decarbonization goals set in the Paris agreement. Many domestic and specialized applications require clean rooms (indoor built environments) for safe and clean operation. Energy efficiency in clean room spaces depends on maintaining livable or required conditions such as temperature, humidity, and particle concentration with minimal use of energy and new carbon dioxide (CO2) emissions. In the literature, parameters such as temperature, relative humidity, particle concentrations, and CO2 emissions are not able to be properly controlled in clean room systems. The designed system in the literature involves high energy consumption and high economic costs. All these factors add novelty to this research, which was a significant research gap in previous studies. This clean room is directly linked to environmental parameters such as ambient temperature, relative humidity, etc. The clean room is also related directly to the building and infrastructure in such a way that there are certain regulatory requirements for designing a clean room. For designing and constructing the controlled environment in a clean room, the English (EN) documents, ISO 9000, and various other standards allow for clean rooms for different types of products. In this research, the designed control configurations properly control the system. Additionally, this system is energy efficient, with positive environmental aspects regarding CO2 emissions. Three control configurations were designed in this research, option A, option B, and option C, and three parameters are controlled in the study. These parameters are room temperature, relative humidity, and CO2 emissions (outside the room). CO2 emissions are controlled outside the room (in the environment). In the last research phase, a comparative analysis of these three control configurations was performed to find an energy-efficient system with fewer CO2 emissions. Control configuration B (option B) provides reliable results regarding an energy-efficient system and fewer CO2 emissions emitted to the environment. In this study, an optimized configuration for the air conditioning system was developed for a clean room (volume 185.6 m3) with a required temperature of 23 °C, relative humidity of 40%, and a particle size of less than 0.3 μm. Three different design configurations were analyzed using TRNSYS simulation software. The minimization of energy use and CO2 emissions were the objective functions. Energy loads were calculated for each of the configurations by varying the fixed air change per hour and the minimum outdoor air flow rate. The results of a whole year simulation run for control configurations A, B, and show that, on the one hand, the ambient weather conditions of temperature and relative humidity (RH) is varied throughout year and, on the other hand, the clean room temperature was maintain at exactly 23 °C, which is the required set point temperature, for all the three configurations (A, B, and C). Furthermore, the clean room relative humidity was maintained at 36% for configuration A, below the 40% which was the set point for clean room relative humidity, and at 40% for configurations B and C. Configuration B exhibited the minimum energy use (7300 kWh), at a fixed air change per hour value of 20 and a minimum outdoor air flow rate of 150 L/s, with the least amount of CO2 emissions, offering an overall 25% improvement over configurations A and C.