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Background This single-center retrospective study aims to analyze the clinical characteristics, treatment strategies, and outcome at discharge of neonatal-onset herpes simplex virus encephalitis (NHSE). Methods We conducted a single-center retrospective case review of infants diagnosed with NHSE at the Children's Hospital of Fudan University between February 1, 2016, and February 1, 2024. Clinical data, including demographics, clinical symptoms, laboratory findings, neuroimaging results, treatment regimens, and outcomes at discharge, were collected and analyzed. Results A total of 14 infants with NHSE (7 males, 7 females) were identified at our center, with a median age at diagnosis of 26 days (range: 7–51 days). Initial symptoms predominantly included fever and seizures, with neurological involvement (e.g., seizures, lethargy, irritability or altered mental states) in 13 cases. Physical examinations, such as bulging anterior fontanel, were noted. Herpes simplex virus (HSV)-DNA was detected in 13 cases (6 HSV-1, 7 HSV-2) through cerebrospinal fluid (CSF) polymerase chain reaction (PCR) or metagenomic testing. Among these, 9 cases were identified via CSF-PCR, with 7 testing positive on the initial examination and 2 on repeated testing. Notably, 6 cases were diagnosed using metagenomic next-generation sequencing (mNGS), all of which yielded positive results on the first test. Ten out of the 12 children often exhibited temporal lobe spikes on video electroencephalograms (VEEGs). Early magnetic resonance imaging (MRI) revealed cytotoxic edema, progressing to multicystic encephalomalacia. All received acyclovir antiviral treatment. Seven discontinued treatments, one was referred for ocular lesions, and six improved and were discharged. Conclusions In this single-center cohort, NHSE often presents with nonspecific fever and seizures, with late onset and absent indicative rashes, complicating early diagnosis. For newborns suspected of having NHSE, early CSF HSV-DNA testing and prompt antiviral treatment are essential to improve outcomes. Metagenomic sequencing is especially valuable for accurate, rapid diagnosis when conventional methods fail.