Ozone is a powerful oxidant capable of killing insects and microorganisms, and eliminating odors, taste, and color. Thus, it could be useful as a fumigant to decontaminate honey comb between uses. The experiments here are intended to determine the exposure levels required to kill an insect pest and spore forming bee pathogens. Ozone was effective against greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), even on naturally infested comb. Neonates and adults were the easiest life stages to kill, requiring only a few hours of exposure, whereas eggs required a 48-h exposure (at 460–920 mg O₃/m³). Two honey bee, Apis mellifera L. (Hymenoptera: Apidae), pathogens, Ascosphaera apis (a fungus that causes chalkbrood) and Paenibacillus larvae (a bacterium that causes American foulbrood), also were killed with ozone. These pathogens required much higher concentrations (3,200 and 8,560 mg O₃/m³, respectively) and longer exposure periods (3 d) than needed to control the insects. P. larvae was effectively sterilized only when these conditions were combined with high temperature (50°C) and humidity (≥75% RH). Thus, ozone shows potential as a fumigant for bee nesting materials, but further research is needed to evaluate its acceptability and efficacy in the field. The need for a reliable method to decontaminate honey bee nesting materials as part of an overall bee health management system is discussed.