Disinfectant application to gloved hands before handling SPF mice is standard practice to minimize transmission of pathogens and microbial contamination between cages. The risk of contamination with murine pathogens on gloves as well as the efficacy of disinfectant application for this step is largely unknown. This study aimed to determine if murine norovirus (MNV), Helicobacter spp., and Rodentibacter spp. are detectable on gloved hands and, if they are, to evaluate how effective the application of a hydrogen peroxide-based disinfectant (Rescue) or 70% ethanol is in reducing the transfer of these pathogens while handling multiple cages of mice. Mice with natural infections of these pathogens were handled without the application of any disinfectant and the gloves were swabbed for PCR testing. All pathogens were detected via PCR with Helicobacter spp. the most frequently transferred in 83% of the cages handled. The mice were then divided into 4 treatment groups based on the product applied to gloves before handling: Rescue, 70% ethanol, sterile water, and no product. Mice in each cage were briefly handled, and the gloves were swabbed with ATP swabs after each cage and swabs for PCR testing after handling 4 and 9 cages, consecutively. All pathogens were detected via PCR in all treatment groups, and neither Rescue nor 70% ethanol was superior to water or no product in reducing contamination. Rescue and 70% ethanol were effective in maintaining lower levels of organic microbial contamination than water and no product for consecutive handling of up to 3 and 4 cages of mice, respectively. This study indicates that exposure to MNV, Helicobacter spp., and Rodentibacter spp. from handling mice is a risk and the application of Rescue or 70% ethanol is not completely effective in eliminating transfer of these pathogens.