Honeybees (Apis mellifera) play a critical role in agricultural pollination. However, their health and numbers are in decline. A major cause of this decline is bacterial diseases, of which American foulbrood disease (AFB) is particularly important and troubling. Since the causative agent, Paenibacillus larvae, is spore forming, it can resist antibiotics, many disinfectants, and environmental stresses. We provide protocols and methods for the growth, maintenance, sporulation, and germination of P. larvae. Also, this study investigates the sporicidal activity of ClO2 on P. larvae spores. The gas efficacy depends on treatment time and gas level. The effective level was 645-811 ng/ml ClO2 for 30 min, 191-198 ng/ml for 1 hour, 21-18 ng/ml for 2 h and 7-16 ng/ml ClO2 for 4 h. For decontamination of contaminated surfaces, 214- 245 ng/ml ClO2 for 1 h and 191- 200 ng/ml ClO2 for 2 h completely inactivate the spores.