The aim of this study was to evaluate viral DNA load in paediatric patients with herpes simplex infections of the central nervous system. A real-time PCR assay for herpes simplex types 1 and 2 was developed for this 8-year retrospective study that included children with herpes simplex infection of the central nervous system (CNS) admitted to a paediatric hospital in Barcelona. Ten patients were diagnosed with herpes simplex CNS disease, 9 with herpes simplex encephalitis and 1 with neonatal herpes. The rate of the annual incidence of disease was stable during the study period, with a mean rate of 0.8 episodes per 100,000 children per year. Viral load was correlated with the increase in patient age (p < 0.05) and with levels of more than 100 leukocytes/mm3 in CSF p = 0.02. Viral load was correlated with timing of the test in relation to the onset of clinical illness: 1.5 × 104 GE/ml when the test was carried out in the first 72 h versus 7.7 × 106 GE/ml when test was carried out after 5 days of symptoms, p = 0.01. In conclusion, quantitative real-time PCR provides a rapid, easy and accurate diagnosis of herpes virus CNS disease including viral load as supplementary information. Further studies are needed to confirm the relationship between viral load and other clinical, pathological, and genetic variables.