Interferon alpha (IFN alpha) is commonly used for the treatment of chronic hepatitis B (CHB) patients. There are 13 different IFN alpha subtypes in humans, but only the subtype IFN alpha 2 is used for clinical treatment. The antiviral activities of all other IFN alpha subtypes against HBV have not been studied. To obtain basic knowledge about the direct antiviral as well as the immunomodulatory effects of IFN alpha subtypes, we used the HBV hydrodynamic injection (HI) mouse model. Application of most IFN alpha subtype proteins inhibited HBV replication in vivo, with IFN alpha 4 and IFN alpha 5 being the most effective subtypes. Decreased viral loads after therapeutic application of IFN alpha 4 and IFN alpha 5 correlated with expanded effector cell populations of NK cells and T cells in both liver and spleen. Hydrodynamic injection of plasmids encoding for the effective IFNa subtypes (pIFN alpha) was even more potent against HBV than injecting IFNa proteins. The combination of pIFN alpha 4 and pIFN alpha 5 showed a synergistic antiviral effect on HBV replication, with a strong increase in NK cell and T cell activity. The results demonstrate distinct anti-HBV effects of different IFN alpha subtypes against HBV in the mouse model, which may be relevant for new therapeutic approaches.