This study was designed to evaluate the efficacy and safety of a novel free polyaggregated amphotericin B (FPA) formulation used to treat experimental canine leishmaniosis (CanL) caused by Leishmania infantum. Eight healthy beagles were intravenously challenged with 5 × 107 promastigotes per mL of L. infantum. One year after infection, they received an intravenous dose of FPA (5mg/kg) every 2 weeks three times.Dogs were assessed monthly for clinical signs, serology, and parasite detection during a followup period of 6 months. Transient adverse effects (i.e., hypotension, diarrhea, bodyweight loss, fever, and asthenia) were observed within 24-48 hours after treatment in 4 animals. In three dogs mean clinical signs scores were reduced. Antibody titers measured by immunofluorescence antibody test (IFAT) had significantly diminished at the end of the study, although according to bone marrow smears and cultures a high percentage of dogs tested positive for the parasite at 6 months posttreatment (PT6). Real-time quantitative PCR (rtQ-PCR) on blood, bone marrow, and urine samples revealed the presence of parasitic DNA in all animals at PT6, although blood loads of the parasite were reduced. These findings indicate that FPA at the dosing regimen used did not achieve clinical or parasitological cure in dogs experimentally infected with L. infantum.