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Antimicrobial Resistance (AMR) is a global health issue with profound implications for human health, economy, and agriculture worldwide. Maternal colonization with antibiotic-resistant bacteria serves as a surrogate marker for the prevalence of such bacteria in the community and also gives a clue to the likely bacteria that cause early onset neonatal sepsis. This study aims to determine the proportion of colonization with selected antibiotic-resistant bacteria, namely Methicillin Resistant Staphylococcus aureus (MRSA) and Extended spectrum beta-lactamase (ESBL) -producing E. coli, Klebsiella, and Enterobacter species. This was a descriptive cross-sectional study that involved 235 pregnant mothers admitted for delivery at the Teaching Hospital, Batticaloa, in the Eastern Province, Sri Lanka, from May to August 2023. Self-collected nasal, rectal, and vaginal swabs were used. Rectovaginal MRSA and Enterobacteriaceae colonization were assessed using separately collected rectal and vaginal swabs processed together. Staphylococcus aureus, MRSA and Enterobacteriaceae were identified using routine microbiological methods and disc-diffusion antibiotic sensitivity testing (ABST). Polymerase Chain Reaction (PCR) was performed to detect the resistance genes in phenotypically identified MRSA and ESBL -producing Enterobacteriaceae isolates. Maternal factors associated with colonization and neonatal outcomes associated with colonization were also identified. Among 235 pregnant women, MRSA colonization was detected in 33 (14.0%), with 36 isolates from multiple sites. Nasal and rectovaginal MRSA colonization was detected in 12 (5.1%) and 24 (10.2%), respectively. ESBL-producing E. coli and Klebsiella species were identified in 9 (3.8%) and 1 (0.4%). All MRSA isolates were sensitive to vancomycin, with high erythromycin resistance (88.9%). Escherichia coli, Klebsiella spp, and Enterobacter spp showed > 70% susceptibility to the tested antibiotics. mecA was the predominant MRSA resistance determinant, and blaCTX-M was the most common ESBL gene. Anaemia in pregnancy was significantly associated with MRSA colonization (p < 0.001) in univariate analysis. Maternal and neonatal outcomes revealed no significant associations. In this study, we found that the prevalence of maternal colonization with MRSA was 14.0%, while ESBL-producing selected Enterobacteriaceae (Escherichia coli, Klebsiella spp, and Enterobacter spp) was 4.3%. The study findings contribute to the growing body of evidence needed for regional planning and prioritization in antimicrobial resistance surveillance and infection prevention in maternal healthcare programs.