The Zika virus (ZIKV) and poxvirus infection are considered as public health emergencies, necessitating the development of effective vaccines. Here, we report novel recombinant DNA-based and non-replicating vaccinia virus (NTV)-based vaccine candidates that express the precursor membrane-envelope (prME) or envelope (E) glycoproteins of ZIKV. After immunization of BABL/c mice with the vaccines using a homologous protocol (DNA/DNA, NTV/NTV) or heterogeneous (DNA/NTV) protocol, a similar level of anti-E IgG and neutralizing antibodies (microneutralization test) were detected in the mice. However, a significantly higher level of E-specific T cell responses was elicited in mice when a heterogeneous prime-boost protocol was used (DNA/NTV) with either the DNA-based or NTV-based vaccines. Furthermore, neutralizing antibodies and a T cell immune response against the vaccinia virus (VV) were detected in mice that were subjected to the prime-boost protocol (DNA/NTV), whereas those subjected to a homologous NTV/NTV protocol had higher levels of anti-VV IgG and neutralizing antibodies. Lastly, a novel H-2d-restricted CD8 T-cell epitope, VRSYCYEASISDMAS, was identified in the ZIKV E protein. These data demonstrate proof of concept of a bivalent vaccine candidate against ZIKV and orthopoxvirus, and support the use of DNA-prME prime and NTV-E boost protocols to protect against ZIKV and orthopoxvirus infections.