Effective control of spore-forming bacilli begs suitable physical or chemical methods. While many spore inactivation techniques have been proven effective, electron beam (EB) irradiation has been frequently chosen to eradicateBacillusspores. Despite its widespread use, there are limited data evaluating the effects of EB irradiation onBacillusspores. To study this,B. atrophaeusspores were purified, suspended in sterile, distilled water, and irradiated with EB (up to 20 kGy). Irradiated spores were found (1) to contain structural damage as observed by electron microscopy, (2) to have spilled cytoplasmic contents as measured by spectroscopy, (3) to have reduced membrane integrity as determined by fluorescence cytometry, and (4) to have fragmented genomic DNA as measured by gel electrophoresis, all in a dose-dependent manner. Additionally, cytometry data reveal decreased spore size, increased surface alterations, and increased uptake of propidium iodide, with increasing EB dose, suggesting spore coat alterations with membrane damage, prior to loss of spore viability. The present study suggests that EB irradiation of spores in water results in substantial structural damage of the spore coat and inner membrane, and that, along with DNA fragmentation, results in dose-dependent spore inactivation.