One concern with traditional smallpox vaccination is inadvertent spread of virus to atopic or immunocompromised contacts. To reduce this risk, we tested the ability of povidone iodine to inactivate infectious virus at the vaccination site beginning at 7 days after transcutaneous smallpox vaccination. This ointment rapidly inactivated virus on the skin without reducing neutralizing antibody titers or antiviral T cell responses. Moreover, there was no delay in healing/eschar separation following povidone iodine application. Together, this indicates that administration of an antiviral/antimicrobial cream can effectively block virus shedding after traditional smallpox vaccination and reduce the risks of autoinoculation or contact spread.