Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, caused by Paracoccidioides spp. A limited number of antifungal agents are available and the search for new compounds has increased. Additionally, nanostructured lipid system (NLS) has emmerged as an interesting strategy to carrier compounds for the treatment of mycosis. In this work, the antifungal efficacy and toxicity of dodecyl gallate (DOD) associated with a NLS was evaluated through in vitro and in vivo tests. DOD showed good in vitro antifungal activity and low toxicity in lung fibroblasts and zebrafish embryos, but no antifungal efficacy in infected mice, which may have been a result of low bioavailability. On the other hand, the association of DOD + NLS was beneficial and resulted in lower toxicity in lung fibroblasts and zebrafish embryos. In addition, NLS + DOD promoted a significant reduction in the fungal burden of mice lungs and could be a potential therapeutic option against PCM.