Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using and the expression of antimicrobial peptide genes in . None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against spp. The peptides could induce an immune response in . Moreover, the peptides caused a delay in the death of spp. infected larvae. Regarding , the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings.