Cytomegalovirus (CMV) infection is a significant complication in paediatric haematopoietic stem cell transplant (HSCT), with substantial morbidity and mortality. While pre-emptive treatment guided by CMV viral load thresholds helps prevent end-organ disease, paediatric protocols have largely been extrapolated from adult populations without robust validation. This prospective interventional study aimed to determine an optimal viral load threshold for initiating pre-emptive CMV therapy in paediatric HSCT patients and test the impact of this threshold in real-world practice. Initially, 219 paediatric HSCT patients from 2009 to 2016 were considered, with weekly CMV quantitative polymerase chain reaction (qPCR) monitoring and treatment initiation at 2500 IU/mL. A mathematical model was developed to analyse CMV kinetics, morbidity and mortality, suggesting a threshold of 1000 IU/mL would be most suitable to prevent adverse outcomes. Subsequently, the new treatment threshold was implemented, and outcomes were then compared with a second cohort of 344 patients treated under the new guidelines from 2017 to 2021. There were significant reductions in CMV viral loads, CMV-associated end-organ disease (27/117, 23.1% vs. 4/98, 4.1%) and mortality (36/117, 30.8% vs. 19/98, 19.4%). These findings support a new evidence-based viral load threshold of 1000 IU/mL or less for pre-emptive treatment in paediatric HSCT recipients to optimise clinical outcomes.