Effective disinfection of hospital rooms after discharge of patients with Clostridium difficile infection (CDI) is necessary to prevent transmission. Unfortunately, several studies have demonstrated that it is not uncommon for environmental cultures to remain positive for C. difficile after cleaning and disinfection of rooms in which a patient with CDI has been hospitalized (CDI rooms) by environmental services personnel. Cultures for C. difficile could potentially be useful to monitor disinfection of CDI rooms, but they are neither widely available nor efficient. There is a need for easy-to use and rapid methods to assess the effectiveness of CDI room disinfection. Adenosine triphosphate (ATP) bioluminescence assays provide a rapid assessment of cleaning effectiveness, because detection of ATP on surfaces indicates the presence of residual organic material (eg, bacteria, human secretions or excretions, and food). Detection of ATP is commonly used in the food and beverage industry and is increasingly being used in health care facilities to assess the adequacy of cleaning procedures. It is not known whether measurement of ATP on surfaces is useful to evaluate disinfection of CDI rooms. Here, we tested the hypothesis that low ATP readings on cleaned surfaces in CDI rooms would be predictive of negative cultures for C. difficile.