The development of biocompatible polymeric nanoparticles has become an important strategy for optimizing the therapeutic efficacy of many classical drugs, as it may expand their activities, reduce their toxicity, increase their bioactivity and improve biodistribution. In this study, nanoparticles of Amphotericin B entrapped within poly (lactic-co-glycolic) acid and incorporated with dimercaptosuccinic acid (NANO-D-AMB) as a target molecule were evaluated for their physic-chemical characteristics, pharmacokinetics, biocompatibility and antifungal activity. We found high plasma concentrations of Amphotericin B upon treatment with NANO-D-AMB and a high uptake of nanoparticles in the lungs, liver and spleen. NANO-D-AMB exhibited antifungal efficacy against Paracoccidioides brasiliensis and induced much lower cytotoxicity levels compared to D-AMB formulation in vivo and in vitro. Together, these results confirm that NANO-D-AMB improves Amphotericin B delivery and suggest this delivery system as a potential alternative to the use of Amphotericin B sodium deoxycholate. Biodistribution scintigraphic images of nanoparticles labeled with the radioisotope technetium 99: amphotericin loaded nanoparticles (99mTc-NANO-D-AMB) or free DMSA after 1 and 8 h of intravenously injection. Display omitted •Amphotericin B is a widely used antifungal, but toxicity limits its use.•NANO-D-AMB was proposed to optimize efficacy and reduce toxicity of this drug.•NANO-D-AMB had similar antifungal efficacy than AMBISOME®.•NANO-D-AMB had lower toxicity compared to D-AMB in vivo and in vitro.•NANO-D-AMB improves drug delivery and might be a potential alternative to D-AMB.