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Background: Group B Streptococcus (GBS) colonization is one of the major causes of severe neonatal infections. The study was intended to identify GBS colonization in pregnant women, explore its potential risk factors, and analyze the impact of GBS on outcomes for both mothers and newborns. Material and Methods: A retrospective research was carried out on pregnant women who had undergone GBS screening and delivered from June 2020 to December 2022. Pregnant women between 35 and 37 weeks of gestation had GBS screening using real-time polymerase chain reaction (RT-PCR). The clinical characteristics and outcomes of mothers and newborns were collected. The risk factors linked to maternal GBS colonization and its impact on adverse outcomes for mothers and neonates were assessed using chi-square and logistic regression analyses. The composite neonatal adverse outcomes included low Apgar scores, neonatal pneumonia, neonatal hyperbilirubinemia, neonatal sepsis, or low birth weight. Results: Overall, the rate of GBS positivity was 10.63% (551/5183), and the rate of maternal GBS screening was 88.4%. Diabetic pregnant women were more likely to become colonized with GBS. Our research revealed that GBS carriers experienced higher rates of fetal distress and neonatal adverse outcomes than non-GBS carriers. Fetal distress (OR, 1.940; 95% CI, 1.355 to 2.778, P < 0.001), neonatal sepsis (OR, 5.063; 95% CI, 2.536–10.109, P < 0.001), low Apgar scores (OR, 2.097; 95% CI, 1.184–3.715, P = 0.011), neonatal pneumonia (OR, 1.638; 95% CI, 1.039 to 2.582, P = 0.034) and neonatal hyperbilirubinemia (OR, 1.438; 95% CI, 1.080 to 1.915, P = 0.013) were significantly related to maternal GBS colonization. When we used the composite adverse neonatal outcomes as the dependent variable and analyzed the influencing factors, the logistic regression analysis revealed that GBS colonization was still significantly related to an elevated risk of adverse neonatal outcomes (OR = 1.752, 95% CI, 1.389–2.208; P < 0.001). Conclusions: Diabetes may be a risk factor for maternal GBS colonization. Moreover, in this study, GBS colonization correlated with neonatal adverse outcomes but not with maternal outcomes. [ABSTRACT FROM AUTHOR]