Epstein-Barr Virus (EBV) is the causative agent of acute infectious mononucleosis and associates with malignancies such as Burkitt lymphoma, nasopharyngeal carcinoma, and non-Hodgkin's lymphoma. Additionally, EBV is responsible for B-lymphoproliferative disease in the context of HIV-infection, genetic immunodeficiencies and organ/stem-cell transplantation. Here we discuss past and current efforts to design an EBV vaccine. We further describe preliminary studies of a novel cocktail vaccine expressing both lytic and latent EBV proteins. Specifically, a tetrameric vaccinia virus (VV) -based vaccine was formulated to express the EBV lytic proteins gp350 and gp110, and the latent proteins EBNA-2 and EBNA-3C. In a proof-of-concept study, mice were vaccinated with the individual or mixed VV. Each of the passenger genes was expressed in vivo at levels sufficient to elicit binding antibody responses. Neutralizing gp350-specific antibodies were also elicited, as were EBV-specific T-cell responses, following inoculation of mice with the single or mixed VV. Results encourage further development of the cocktail vaccine strategy as a potentially powerful weapon against EBV infection and disease in humans.