Amphotericin B (AmB) is widely applied in treatment of systemic fungal infections. However, the emergence of severe adverse effects, such as nephrotoxicity, hepatotoxicity and hemolytic anemia, can limit its clinical use. Poly(lactide-co-glycolide) (PLGA) or poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) blend nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. Nanoparticles were characterized by particle size, polydispersity index, Fourier transform infrared spectroscopy, differential scanning calorimetry and X-ray diffraction analyses. The antifungal activity was evaluated against strains of Candida albicans and Cryptococcus neoformans. Toxicity was evaluated by hemolysis assay and after 7 days treatment in rats. Mean nanoparticle size was below 200nm with low polydispersity and AmB encapsulation efficiency higher than 90%. Nanoencapsulation resulted in AmB amorphization and no chemical interaction between drug and polymer. In C. albicans, minimum inhibitory concentration (MIC) of AmB-loaded PLGA-PEG nanoparticles was 2-fold higher than free AmB or marketed deoxycholate AmB (AmB-D), while MIC of AmB-loaded PLGA nanoparticles was 10-fold higher than AmB-loaded PLGA-PEG. In C. neoformans, the efficacy of AmB-loaded PLGA nanoparticles was comparable to free AmB, while AmB-loaded PLGA-PEG nanoparticles and AmB-D did not present MIC in tested concentration range. Nanoparticles inhibited the AmB-induced hemolysis. After 7 days treatment in rats, histologic examination revealed AmB-D treatment presented initial liver damage, while AmB-loaded nanoparticles did not present any hepatic cellular alteration. Kidney alteration was not observed in all treatments. Thus, PLGA and PLGA-PEG nanoparticles are promising carriers for AmB delivery with potential application in antifungal therapy.