Objective: To estimate the incidence of neonatal herpes simplex virus (HSV) infection and the number of neonates with suspected invasive bacterial infection (IBI) needed to treat (NNT) with acyclovir to ensure prompt treatment of invasive HSV infections. Design: A nationwide population-based cohort study. Setting: All neonatal and paediatric emergency departments in Denmark from 1 January 2010 to 31 December 2019. Patients: Neonates aged 0-28 days with HSV infection. Main outcome measures: The main outcome measures were incidence and NNT. The NNT was calculated based on neonates with invasive HSV infection whose onset symptoms resembled IBI and the estimated number of Danish neonates who received antibiotics for suspected IBI. Results: Fifty-four neonates with HSV infection were identified, that is, an incidence of 9 per 100 000 live births. Twenty presented with symptoms resembling IBI, all within the first 14 days of life. Of 18 (78%) neonates, 14 had elevated C reactive protein, 14 of 19 (74%) had elevated alanine aminotransferase and 11 of 17 (65%) had thrombocytopaenia. The estimated NNTs with empiric acyclovir at postnatal ages 0-3, 4-7 and 8-14 days were 1139 (95% CI 523 to 3103), 168 (95% CI 101 to 726) and 117 (95% CI 48 to 198), respectively. Conclusions: The incidence of neonatal HSV infection was higher than in previous decades; however, the estimated NNT with empiric acyclovir was high. Therefore, we propose not to treat all neonates suspected of IBI with empiric acyclovir, as current European guidelines suggest. However, HSV should be considered in neonates with signs of infection, especially after the third postnatal day and in neonates with high alanine aminotransferases and thrombocytopaenia.