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Mycoplasma is minute bacteria that can be found ubiquitously in the environment and also in human, animal and plant tissues. In addition to their public health importance as aetiological agents of infections and possible association with certain cancers, mycoplasma is a major contamination concern in biotechnology. These bacterial cells are very small, can form biofilms and survive for extended periods of time when dried onto surfaces. Despite these concerns, there is little information concerning their resistance to currently used disinfection methods. The objective of this study was to evaluate commonly used biocidal treatments against three representative mycoplasma species.Mycoplasma was dried onto stainless steel coupons and exposed to decontamination products. All strains survived drying and any significant viability loss because of the test method (including neutralization), as demonstrated by a ≤0·5 log10 for each tested species. The quaternary ammonium compound (QAC) tested presented poor efficacy, whereas 70% ethanol was fully efficient with complete inactivation after 5-min exposure. Alkaline cleaner formulations presented increasing efficacy when tested at 0·2, 0·4 and 0·8% concentrations, with complete kill observed at 0·8% of two products tested. Decontamination with vaporized (gaseous) hydrogen peroxide (VHP) was very efficient at concentrations used for room and small enclosures decontamination (180-1200 ppm with various time exposures), as well as for device sterilization applications.Ethanol and alkaline detergent formulations were particularly efficient against mycoplasma, but a QAC formulation was not. VHP in room disinfection and device sterilization applications was effective against all mycoplasma species tested.Mycoplasma can provide resistance to environmental factors (such as drying) and disinfectants. Further studies are required to confirm the effectiveness of other disinfectants and the mechanisms of mycoplasma resistance.