Objective To evaluate the performance of metagenomic next-generation sequencing (mNGS) in detecting pathogens in suspected neonatal sepsis and central nervous system infections. Methods This retrospective study included 648 neonates with suspected sepsis or central nervous system infections, with 734 cerebrospinal fluid and 733 blood samples collected. The pathogen spectra detected by mNGS and traditional culture were compared. Using clinical diagnosis as the gold standard, the diagnostic efficacy of the two methods was analyzed. Results The positive rates of pathogen detection by mNGS in cerebrospinal fluid and blood samples were 15.3% and 40.0%, respectively, significantly higher than those of traditional culture (1.4% and 10.7%, respectively). mNGS identified 25 and 40 distinct pathogenic species from cerebrospinal fluid and blood, respectively, exceeding the 4 and 24 species detected by culture. Ureaplasma, Mycoplasma, and other fastidious pathogens difficult to culture were detected exclusively by mNGS. Using clinical diagnosis as the reference, mNGS showed sensitivities of 50.4% (cerebrospinal fluid) and 46.7% (blood), compared to 5.8% and 18.0% for culture. Conclusions mNGS significantly improves pathogen detection rates in neonatal infections compared with traditional culture, provides more comprehensive pathogen information, and holds important clinical value for the precise diagnosis and treatment of neonatal infections.