Gastrointestinal infections represent a significant global health burden, ranking as the second leading cause of mortality among infants and children. Identifying of pathogens causing neonatal gastrointestinal infections has presented tough challenges. This study aimed to summarize the prevalence of common pathogens associated with neonatal gastrointestinal infections through a comprehensive systematic review and meta-analysis of published literature. The last search was performed on January 08, 2025, from databases including EMBASE, PubMed, Cochrane Libary, and Web of Science. The outcome variable was infection rate, and the detection methods used were blood culture, tissue culture, or molecular biology methods. Two researchers independently extracted the research data and evaluated its quality using the JBI Critical Appraisal Tools. Twenty-three studies met the inclusion criteria. The pooled prevalence rates of common pathogens were as follows: Bacteria, including Escherichia (22.2%; 95% CI 8.3-40.4%, I2 = 98%), Clostridium (21.8%; 95% CI 2.2-53.8%, I2 = 96%), Klebsiella (19.2%; 95% CI 8.3-33.4%, I2 = 97%), Staphylococcus (13.6%; 95% CI 6.0-23.7%, I2 = 91%), Enterococcus (12.4%; 95% CI 1.8-30.3%, I2 = 96%), and Streptococcus (6.8%; 95% CI 2.5-12.9%, I2 = 43%). Fungi, including Candida (3.8%; 95% CI 0.6-9.6%, I2 = 84%). Viruses, including Rotavirus (11.6%; 95% CI 1.0-31.5%, I2 = 94%) and Adenovirus (4.1%; 95% CI 0.5-11.0%, I2 = 58%). Peritoneal culture methods demonstrated significantly higher positivity rates compared to other detection methods. Escherichia coli exhibited consistently high positivity rates across the three main detection methods. Klebsiella showed the highest positivity rates among bacterial isolates in both blood and peritoneal cultures. Pathogen detection and prevalence in necrotizing enterocolitis (NEC) cases were markedly higher compared to other conditions. This meta-analysis identifies key pathogens in gastrointestinal infections, including Klebsiella pneumoniae, Escherichia coli, Candida, Rotavirus, Adenovirus, and others that are suspected before clinical sample results are available. It also highlights that intestinal pathogen infections are linked to an increased risk of neonatal necrotizing enterocolitis (NEC) and emphasizes the advantages of peritoneal culture in detecting these infections.© 2025. The Author(s).