BACKGROUND: In low-income countries, data on prevalence and effects of group B Streptococcus (GBS) and Escherichia coli (E. coli) colonization among pregnant women are scarce, but necessary to formulate prevention strategies. We assessed prevalence of GBS and E. coli colonization and factors associated among pregnant women, its effect in newborns and acceptability regarding the utilized sampling methods in a semirural Mozambican hospital. METHODS: Pregnant women were recruited from June 2014 to January 2015, during routine antenatal clinics at gestational age ≥ 34 weeks (n = 200); or upon delivery (n = 120). Maternal risk factors were collected. Vaginal and vagino-rectal samples for GBS and E. coli determination were obtained and characterized in terms of antimicrobial resistance and serotype. Anti-GBS antibodies were also determined. Neonatal follow-up was performed in the first 3 months after birth. Semistructured interviews were performed to investigate acceptability of sample collection methods. RESULTS: In total, 21.3% of women recruited were GBS carriers, while 16.3% were positive for E. coli. Prevalence of HIV was 36.6%. No association was found between being colonized by GBS and E. coli and maternal risk factors. GBS isolates were fully susceptible to penicillin and ampicillin. Serotypes V (32.4%), Ia (14.7%) and III (10.3%) were the most commonly found and 69.2% of the women tested had immunoglobuline G antibodies against GBS. E. coli isolates showed resistance to ampicillin in 28.9% and trimethoprim/sulfamethoxazole in 61.3% of the cases. CONCLUSION: Prevalence of GBS and/or E. coli colonization among pregnant women is high in this semirural community and comparable with those reported in similar settings. Four serotypes accounted for nearly 70% of all isolates of GBS. Population-based data on infant GBS infections would enable the design of prevention strategies for GBS disease in Mozambique.