Liposomal amphotericin B (Ambisome) is the gold standard for the treatment and prevention of fungal infections both in the adult and pediatric populations. The lyophilized dosage form has to be reconstituted and diluted by hospital staff, but its management can be challenging due to the spontaneous tendency of amphotericin B to form aggregates with different biological activity. In this study, the colloidal stability of the liposomes and the chemical stability of amphotericin B were investigated over time at storage conditions. Three liposomal formulations of amphotericin B at 4.0 mg/mL, 2.0 mg/mL, and 0.2 mg/mL were prepared and assayed for changes regarding the dimensional distribution, zeta potential, drug aggregation state, and onset of by-products. Our analyses highlighted that the most diluted formulation, kept at room temperature, showed the greatest changes in the aggregation state of the drug and accordingly the highest cytotoxicity. These findings are clinically relevant since the lower dosages are addressed to the more vulnerable patients. Therefore, the centralization of the dilution of AmBisome at the pharmacy is of fundamental importance for assuring patient safety, and at the same time for reducing medication waste, as we demonstrated using the cost-saving analysis of drug expense per therapy carried out at the G. Gaslini children hospital.