Disinfection of air conditioning rooms are related to reduce the risk of pathogenic epidemics. Various chemical and physical disinfection approaches are applied to the rooms for healthcare. In this study, efficacy of ultraviolet radiation on microbial elimination of air conditioning room was analysis. Ultraviolet chamber is constructed and assembled over the air conditioner in 25 cubic meter room. Ultraviolet-C (UVC) is radiated to airflow out of air conditioner. Five places of enrich media were placed on different position of room floor at different time interval for fifteen minutes. Plates were closed and reversed incubation at room temperature. Different types of microbial colonies on plates were counted. Average number of colonies from radiation on airflow room were analyzed and compared to those from non-radiation on airflow room (control). The result showed that ultraviolet radiation on conditioning airflow have efficiency on microbial reduction depending on the expose time. Furthermore, ultraviolet radiation may be effect on microbial cell injury because injured colonies were slowly growing and appearing in enrich media. Therefore, UVC radiation chamber assembled over the air conditioner have benefit on eliminating microorganisms in air conditioning room and will be used in enhancing efficacy on room disinfection for various healthcare purposes.