Objective To explore the feasibility and practicability of metagenomic next-generation sequencing (mNGS) technique in rapid identification of infection sources in hospital-associated cluster infection events. Methods Pharyngeal, anal swabs and blood specimens of 6 neonates with fever and diarrhea (or rash), as well as specimens (pharyngeal and anal swabs) of other neonates and health care workers (HCWs) in the same neonatal center of a hospital during the same period and environmental specimens were collected, traditional culture method, enterovirus universal polymerase chain reaction (PCR), mNGS and Echovirus 18 (ECO18) specific PCR were adopted for detection. Results No pathogen was found in the bacterial culture of 6 neonates' blood, anal and pharyngeal swabs, enterovirus universal PCR and ECO18 specific PCR were both positive for anal swabs. At least one specimen from different specimens of 6 neonates was found ECO18 by mNGS, positive rates from high to low were anal swabs (66.66%), blood (50.00%) and pharyngeal swabs (0). The pharyngeal and anal swab specimens of 18 asymptomatic neonates in the same ward during the same period were detected by mNGS, anal swab specimens of 2 cases were positive for ECO18, HCWs and environmental specimens didn't found ECO18 or other enterovirus. Conclusion mNGS can detect low copy number of ECO18 in feces or serum of patients infected with ECO18, it is an effective auxiliary method for the diagnosis of enterovirus infection, and has the potential to trace the pathogen of hospital cluster enterovirus infection.