Effective cleaning in hospitals is an important aspect of infection control of pathogens such as methicillin resistant Staphylococcus aureus (MRSA). There is a need for simple, rapid methods of assessing cleanliness in order to effectively audit cleaning programmes and to educate staff. The aim of this study was to evaluate the use of adenosine triphosphate (ATP) bioluminescence swabs for assessing cleanliness in hospitals. Sites (n=108) in three hospital wards, including floors, patient equipment and clinical workstations were examined by visual assessment, microbiological swabbing and ATP bioluminescence swabbing. Overall, ATP swabbing detected a similar number of undesirably high results compared to microbiological swabbing, but visual assessment gave significantly fewer unsatisfactory results. Highest median contamination levels (both ATP and microbiological) were obtained from floor sites under patient beds, and the lowest levels from patient equipment. It was concluded that ATP bioluminescence swabbing, while not directly equivalent to microbiological swabbing, was a useful tool for monitoring cleanliness. In addition, because of its ability to produce on-the-spot results, it proved useful during education sessions with ward staff and cleaning staff, as a novel way of demonstrating the importance of cleaning.