Background Primary antifungal prophylaxis in paediatric allogeneic HSCT recipients is mainly based on azoles, which can have related toxicity and drug interactions. Low-dose liposomal amphotericin B (L-AmB) is an attractive intravenous alternative because of its low toxicity and lower risk of interactions. Objectives To evaluate the effectiveness and safety of L-AmB (1 mg/kg/day) for primary antifungal prophylaxis in pre-engraftment paediatric HSCT patients. Patients and methods Retrospective, observational study including all consecutive patients aged ≤18 years who underwent HSCT and received antifungal prophylaxis with intravenous L-AmB (1 mg/kg/day, from day −1 to 48 h before discharge) between January 2012 and December 2016. Results In total, 125 HSCT procedures in 118 patients were included, median age 7.2 years (IQR 4.2–11.5). Haematological malignancies were the main underlying condition (63.6%), and 109 (87.2%) were considered at high risk for invasive fungal infection (IFI). Ten patients (7.7%), all high risk, developed breakthrough IFI (three Candida spp., seven invasive mould infections) and tended to have higher overall mortality. The only statistically significant risk factor for IFI was cytomegalovirus co-infection. Adverse events, all grade I, occurred in 25 (20%), requiring L-AmB withdrawal in one case. Overall survival at 30 days was 99.2%. At study completion, one patient had died of IFI. Conclusions The incidence of breakthrough IFI was comparable to that of previous reports, with a very low rate of significant toxicity. Thus, prophylactic L-AmB may be a safe, effective option for antifungal prophylaxis in the pre-engraftment phase for children undergoing HSCT, even those at high risk.