Aims: To determine the mechanism whereby the new disinfectant Sterilox(R) kills spores of Bacillus subtilis. Methods and Results: Bacillus subtilis spores were readily killed by Sterilox and spore resistance to this agent was due in large part to the spore coats. Spore killing by Sterilox was not through DNA damage, released essentially no spore dipicolinic acid and Sterilox-killed spores underwent the early steps in spore germination, including dipicolinic acid release, cortex degradation and initiation of metabolism. However, these germinated spores never swelled and many had altered permeability properties. Conclusions: We suggest that Sterilox treatment kills dormant spores by oxidatively modifying the inner membrane of the spores such that this membrane becomes non-functional in the germinated spore leading to spore death. Significance and Impact of the Study: This work provides information on the mechanism of spore resistance to and spore killing by a new disinfectant.