Prions pose a challenge to decontamination, particularly before the re-use of surgical instruments. They have relatively high resistance to standard decontamination methods and require extreme chemical and/or heat-based treatments for devices used in known or suspected cases of disease. This study investigated the effectiveness of a new gaseous hydrogen peroxide sterilisation process for prions as an alternative low-temperature method. Gaseous peroxide, in addition to known antimicrobial efficacy, was shown to inactivate prions both in in-vitro and in-vivo assays. In contrast to the gas form, liquid peroxide was not effective. The mechanism of action of gaseous peroxide suggested protein unfolding, some protein fragmentation and higher sensitivity to proteolytic digestion. Hydrogen peroxide liquid showed a degree of protein clumping and full resistance to protease degradation. The use of gaseous peroxide in a standard low-temperature sterilisation process may present a useful method for prion inactivation.