Commercially-reared bumblebees provide an important pollinator service that helps support food production and security. The deployment of an appropriate non-thermal disinfection technology for the bulk treatment of pollen collected from honeybees for the feeding of commercial bumblebees is important in order to mitigate against complex diseases and unwanted pathogen spillover to native bees. High level disinfection of pollen was achieved using an electron (e)-beam dose of 100 kGy that corresponded to 78 % loss of cellular viability of bee pathogens before feeding to bumblebees as measured by the novel in vitro use of flow cytometry (FCM). Novel findings showed that e-beam treated-pollen that was fed to bumblebees produced fewer females, gynes and exhibited an absence of males when compared to control bumblebee colonies that were fed untreated commercial pollen. A similar trend emerged in bumblebee colony reproductive outputs when using membrane filtered washed pollen. Proteomic analysis of bumblebees from individual colonies fed with treated-pollen revealed a differential abundance of proteins associated with stress, immunity and metabolism when compared to the untreated pollen control group. Microbiome analysis of the bumblebee gut content revealed differences in microbiota between treated and untreated pollen in bumblebee colony studies. This novel study evaluated the impact of industrial ebeam treated-pollen on complex bee disease mitigation where physically treated-pollen fed to bumblebees was shown to substantially affect colony reproductive outputs.