Globally, Toxoplasma gondii remains a major One Health challenge, with substantial public health and socioeconomic consequences. Despite its widespread threat, effective control measures are limited by gaps in vaccination strategies. In this study, we experimentally evaluated a Multi-Epitope Vaccine MEV-2A, an in silico-designed multi-epitope recombinant protein incorporating a built-in cholera toxin B subunit as an adjuvant, in addition to B- and T-cell epitopes from seven antigen families. The MEV-2A was expressed, purified, and administered to BALB/c mice (n = 15/group), either alone or with a second adjuvant, either Freund’s or a novel polyethyleneimine-based black seed oil nano-emulsion (PEI/BSO-NE). Specific humoral and cellular immune responses were evaluated, and their protective efficacy was assessed by reduction in parasite DNA burden in peritoneal fluid and survival rate analysis following challenge. The synthesized nano-emulsion (NE) was well dispersed, nano-sized, and had high encapsulation efficiency and pH-responsive release. All three MEV-2A-containing formulations elicited significant specific humoral and cellular immune responses relative to unvaccinated and adjuvant-only controls. They also induced a mixed Th1/ Th2 immune response. Furthermore, they resulted in a notable reduction in parasite DNA burden and prolonged the median survival time. The MEV-2A@Freund’s formulation elicited the highest antibody reactivity, IL-2, and IFN-γ levels, along with 71.38% reduction in parasite DNA burden and median survival of 11 days, whereas the MEV-2A@PEI/BSO-NE induced higher IL-4 and IL-10, 91.28% reduction in parasite DNA burden and median survival of 13 days. These findings identify MEV-2A as a promising vaccine candidate and PEI/BSO-NE as a formulation worth further evaluation. Future studies are warranted to evaluate the protective efficacy of MEV-2A against different genotypes and to comprehensively assess the safety and immunological properties of PEI/BSO-NE as a delivery method and adjuvant platform.