The main focus of this study was to determine the most significant factors that influencing indoor air quality (IAQ) and thermal comfort of a mechanically room in FSB new building. This study measured the concentrations of ozone (O3), nitrogen dioxide (NO2), total volatile organic compound (TVOC), carbon dioxide (CO2), carbon monoxide (CO), particulate matter (PM), temperature and relative humidity using seven set up conditions. The set up condition for laboratory is based on temperatures that have been selected between 22°C to 26°C and window of the room being either close or open. The IAQ were analyzed by descriptive analysis, time series plot and diurnal variations. Furthermore, thermal comfort for the room of FSB new building has been determine by the CBE thermal comfort tool based on ASHRAE 55 standards. Next, multiple linear regression (MLR) was utilized to determine the most significant factors that influencing IAQ and thermal comfort. Result suggested that the IAQ of room FSB new building has maximum value during the window was opened. The setup of selected temperature air conditioner was minimizing the concentrations of IAQ because IAQ were increase during high temperature. Among of the selected IAQ parameters, only particulate matter concentration was surpasses the guideline limit set by DOSH for the indoor environment. The increment of particulate matter concentrations was due to the suspended dust in the room and additional movements of the occupants inside the room exaggerate the condition. Besides, results of this study suggested that a good IAQ inside an air conditioning room was achieved when the temperature is set at 26°C. The result suggested that IAQ parameters such ozone, nitrogen dioxide, total volatile organic compound, carbon dioxide, relative humidity was significant in influencing the room thermal comfort.