The aim of this work was to study the effect of aggregation of amphotericin B (AMB) in their toxicity. The aggregation of AMB depends on different formulation factors such as pH and excipients, therefore three formulations with different AMB aggregation states were prepared: a monomeric form (M-AMB), a dimeric form (D-AMB) and a poly-aggregated form (P-AMB). The predominant aggregation state of each AMB formulation was characterized by spectrophotometry and their size by dynamic laser light scattering. Toxicity was evaluated by lethality in mice and hemolysis test from human erythrocytes and the experimental AMB formulations were compared with reference formulations of AmBisome, Fungizone and heated Fungizone. The less toxic aggregation form of AMB was the poly-aggregated one which was similar to AmBisome. Moreover, the P-AMB and heated Fungizone were centrifuged to isolate different size fractions. The toxicity of these two heterogeneous formulations was related to their size, so the smaller the aggregation size fraction the higher the toxicity determined by hemolysis. It can be concluded that the aggregation state of AMB and their size affects critically the toxicity of AMB. The low toxic P-AMB formulations contain a different poly-aggregated state to that of AmBisome, heated Fungizone and the other studied AMB aggregation states.