Visual inspections are used to assess hospital cleanliness, as visible contamination may present health risks and influence perceptions of care quality. Problematically, many contaminants are invisible to the naked eye, limiting the reliability of visual checks. Many invisible substances, however, fluoresce (i.e. emit visible light after absorbing electromagnetic radiation). Portable torches can detect fluorescent substances in situ, offering a potential method to enhance cleaning practices. This study has evaluated fluorescence as a tool for identifying general invisible contamination after hospital cleaning. Visibly clean surfaces in seven single-occupancy patient rooms and two six-bed wards across two National Health Service hospitals were examined using a portable high-intensity blue and ultraviolet light torch. Adenosine triphosphate (ATP) levels in fluorescent and non-fluorescent areas were taken as a recognized cleaning monitoring tool, and analysed statistically using Wilcoxon signed-rank tests. Fluorescent contamination that was invisible to the naked eye was found on every surface. ATP relative light unit (RLU) levels were significantly higher in fluorescent substances compared with non-fluorescent substances (P≤0.05) with large effect sizes on patient chairs, bed frames, overbed tables, bedside units and pillows, but not toilets, sinks or commodes. The mean RLU measurement was 161 in fluorescent areas compared with 33 RLU in control areas. Fluorescence detected alternative contamination which could present toxic risk to humans, such as cleaning fluid and/or drug-contaminated residue which frequently contain fluorescent constituents. This was an important finding as relying solely on ATP detection may overlook significant contamination risks. Further work to evaluate the method as a cleaning aid is encouraged.