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We sought to explain the unexpected failure of the inorganic copper-based biocide CuWB50 to effectively decontaminate microfibre cleaning cloths that became contaminated with Acinetobacter lwoffii.CuWB50 was diluted using distilled water or tap water obtained from two different ICUs. Microtitre plate assays were used to determine the minimum inhibitory concentration (MIC) for the implicated A. lwoffii. pH and oxidation-reduction potential (ORP) tests were performed and representative water samples were chemically analysed. When diluted in distilled water, the CuWB50 MIC for A. lwoffii was 9 mg l−1 but in tap water from each ICU it was 37 and 75 mg l−1 at hardness levels of 246 and 296 mg CaCO3 l−1 respectively. CuWB50-distilled water solutions consistently had a lower pH and higher ORP than CuWB50-tap water solutions.Hard water adversely affects the biocidal efficacy of CuWB50.Unintentional environmental contamination is a risk when using wet microfibre cloths. This occurred when cloths were stored in CuWB50 overnight combined with the unintentional but erroneous use of tap water. This study emphasizes the need for clearly documented cleaning protocols embedded within a culture of adequate training and constant supervision of cleaning staff.