Orthohantaviruses are rodent-borne pathogens that cause hemorrhagic fever with renal syndrome disease (HFRS) in Eurasia. The highest incidence of orthohantavirus infection is documented in the Volga region of Russia. Puumala orthohantavirus was identified as the causative agent. Life-threatening complications were diagnosed in some HFRS cases leading to prolonged recovery periods or even fatal outcomes. There are limited options for HFRS prevention in Russia. Identifying the most immunogenic epitopes of structural protein is the first stage of vaccine development. This study aimed to evaluate the immunogenicity of a microvesicle (MV)-based vaccine carrying Pummala virus nucleocapsid proteins (PUUV N) or a combination of nucleocapsid and glycoproteins (PUUV N-Gn/Gc). We found the most immunogenic PUUV N peptides after vaccination. Also, we demonstrated that the MV-based vaccine carrying PUUV N or PUUV N-Gn/Gc could elicit cross-protection immunity against N peptides of other orthohantavirus strains.