Aim: To develop nanoemulsions (NEs) loading amphotericin B (AmB) and to evaluate the influence of different excipients on the stability and the supramolecular organization, retentionand toxicity of AmB. Materials & methods: The NEs were developed from different oils, surfactants, external mediaand anionic lipids (disteaoryl phosphatidylglycerol [DSPG]and dioleoyl phosphatidylglycerol [DOPG]). Their impact on the size, pH, zeta potential, AmB encapsulation efficiency, AmB retentionand hemolytic potential of the NEs was evaluated. Results& conclusion: The use of soybean oil (lipid matrix), Span 80 (surfactant), phosphate buffer (external phase) and DSPG or DOPG (hydrophobic ion pair) provided better NE stability, higher AmB retention within the NEs and a safer formulationprofile in hemolysis tests.