Managed honey bee (Apis mellifera) populations are currently facing unsustainable losses due to a variety of factors. Colonies are challenged with brood pathogens, such as the fungal agent of chalkbrood disease, the microsporidian gut parasite Nosema spp., and several viruses. These pathogens may be transmitted horizontally from worker to worker, vertically from queen to egg and via vectors like the parasitic mite, Varroa destructor. Despite the fact that these pathogens are widespread and often harbored in wax comb that is reused from year to year and transferred across beekeeping operations, few, if any, universal treatments exist for their control. In order to mitigate some of these biological threats to honey bees and to allow for more sustainable reuse of equipment, investigations into techniques for the sterilization of hive equipment and comb are of particular significance. Here, we investigated the potential of gamma irradiation for inactivation of the fungal pathogen Ascosphaera apis, the microsporidian Nosema ceranae and three honey bee viruses (Deformed wing virus [DWV], Black queen cell virus [BQCV], and Chronic bee paralysis virus [CBPV]), focusing on the infectivity of these pathogens post-irradiation. Results indicate that gamma irradiation can effectively inactivate A. apis, N. ceranae, and DWV. Partial inactivation was noted for BQCV and CBPV, but this did not reduce effects on mortality at the tested, relatively high doses. These findings highlight the importance of studying infection rate and symptom development post-treatment and not simply rate or quantity detected. These findings suggest that gamma irradiation may function as a broad treatment to help mitigate colony losses and the spread of pathogens through the exchange of comb across colonies, but raises the question why some viruses appear to be unaffected. These results provide the basis for subsequent studies on benefits of irradiation of used comb for colony health and productivity.