Abstract Background In 2000, the World Health Organization (WHO) had eight sets of conflicting recommendations for decontaminating medical equipment. We conducted a systematic review of observational studies to assist WHO in reconciling the various guidelines. This paper summarises the methods developed and illustrates the results for three procedures – alcohol, bleach and povidone iodine. Methods We developed a Medline search strategy and applied inclusion criteria specifying the decontamination procedures of interest and an outcome of microbial destruction for a set of marker organisms. We developed protocols to assess the quality of studies and categorised them according to the reliability of the methods used. Through an iterative process we identified best practice for the decontamination methods and key additional factors required to ensure their effectiveness. We identified 88 published papers for inclusion, describing 135 separate studies of decontamination. Results For disinfection with alcohol, best practice was identified from 23 studies as an exposure to 70–80% ethanol or isopropanol for at least 5 minutes. Bleach was effective for sterilization at a concentration of 5000 ppm for 5 minutes and for disinfection at 1000 ppm for 10 minutes (33 studies). Povidone iodine was only partially effective for disinfection at a concentration of 1% for 15 minutes (15 studies). Conclusions Our findings provide an evidence base for WHO guidelines on decontaminating medical equipment. The results support the recommended use of bleach and show that alcohol could be used more widely than current guidelines suggest, provided best practice is followed. The effectiveness of povidone iodine is uncertain.