Background Portable adenosine triphosphate (ATP)-bioluminometers have been used in the food industry to monitor the effectiveness of surface cleaning but their intended use in hospital infection control suggests a need for instrument validation to confirm effective technology transfer. Methods The performance of three readily available brands of portable bioluminometer was compared in terms of their ability to generate relative light units (RLU) from a range of standard ATP solutions. Quality control of standards was carried out using high pressure liquid chromatography (HPLC). Results There was no significant difference (P≤0.05) in the ability of different meters to effectively measure hygiene change on a log scale in a central measurement range of 0.001 to 1.0mgL-1 ATP. Outside this range meter performance deteriorated, with the possibility of individual and comparative measurement error. No out-of-range warning system existed for any of the meters. While different brands generated widely different log10RLU values for fixed quantities of ATP in this range, curve similarities suggested standardisation possibilities to enable comparison of results. Testing at a higher level of resolution in the 0.0001 to 0.002mgL-1 ATP range proximate to a proposed 100 RLU cleaning benchmark also revealed poor repeatability as a potential for measurement error. Conclusions Portable ATP-bioluminometers, when used to indicate surface cleaning effectiveness, demonstrate reliable performance when measuring over a very wide range of ATP concentrations. Monitoring hygiene in terms of an absolute threshold value such as a cleaning benchmark may, however, be invalid as a concept when using existing portable ATP-bioluminometer technology.