Background: In neonates, early-onset sepsis (EOS) occurring within 72 hours after birth is an important cause of mortality worldwide. Emerging data show that EOS may occur more frequently in tropical and peri-equatorial regions with more gram-negative bacteria than in the Western countries. This systematic review aimed to estimate the prevalence of gram-negative bacteria in the maternal genital tract during the peripartum period. Materials and methods: We explored the primary research studies that reported the presence of gram-negative bacteria in the maternal genital tract using the software STATA, version 17.1. Five databases, PubMed, Embase, Scopus, Web of Science, and ProQuest were searched until October 2022. Data were analyzed using random-effects meta-analyses to determine the prevalence of gram-negative bacteria in the maternal genital tract. Results: Fifteen studies qualified for analysis by our predetermined inclusion criteria. The overall prevalence of gram-negative bacteria in cervical secretions was 23.20% (95% CI [confidence interval]: 11.77–37.08, I²: 99.79%). Escherichia coli (15.3%) and Acinetobacter (0.36%) species reported the highest and lowest prevalent bacteria, respectively. The prevalence of other gram-negative species was Klebsiella pneumoniae (0.47%), Pseudomonas (2.81%), Enterobacter (3.33%), Alcaligenes faecalis (1.32%), Proteus vulgaris (10.0%), and Providencia alcalifaciens (10%). Most of the studies were from tropical countries, and there was a positive linear relationship between the studies. Conclusion: Gram-negative colonization of the maternal cervical-vaginal tract may be more frequent than previously recognized in tropical/peri-equatorial regions of the world. Early identification of these bacterial pathogens may help in timely evaluation and treatment of these infants.