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Samrawit Birhane Fiseha,1 Getamesay Mulatu Jara,2 Elshaday Azerefegn Woldetsadik,3 Fanuel Belayneh Bekele,4 Musa Mohammed Ali2 1Department of Medical Laboratory Science, Hawassa Health Science College, Hawassa, Ethiopia; 2School of Medical Laboratory Science, College of Medicine and Health Sciences, Hawassa University, Hawassa, Ethiopia; 3Department of Microbiology, Hawassa University, Comprehensive Specialized Hospital, Hawassa, Ethiopia; 4School of Public Health, College of Medicine and Health Sciences, Hawassa University, Hawassa, EthiopiaCorrespondence: Musa Mohammed Ali Email ysnmss@yahoo.comIntroduction: Vaginal colonization with some species of bacteria during the last term of pregnancy can affect the health of fetuses and newborns resulting in high morbidity and mortality among newborns.Objective: The aim of this study was to determine the colonization rate of potential neonatal disease-causing bacteria, factors associated with colonization rate, and the antimicrobial susceptibility profile of bacteria among pregnant women.Methods: Institution-based cross-sectional study was conducted on pregnant women from October 13 to December 28, 2020, at government hospitals located in Hawassa, Ethiopia. Background data were captured using a structured questionnaire. Vaginal swabs were collected to isolate bacteria using the standard method. Antimicrobial susceptibility test was performed using the modified Kirby–Bauer disc diffusion method. Data were analyzed using SPSS. Factors that could predict vaginal colonization with potential neonatal disease-causing bacteria were determined using logistic regression.Results: Overall bacterial colonization rate among pregnant women was 271 (98.9%) 95 CI (97.4‒100.1). The prevalence of potential neonatal disease-causing bacteria was 95 (34.7%) 95 CI (28.8‒40.1). The proportion of potential neonatal disease-causing bacteria were as follows: Escherichia coli (n=82, 29.9%), Acinetobacter species (n=9, 3.3%), Staphylococcus aureus (n=7. 2.6%), and Klebsiella pneumoniae (n=4, 1.5%). Pregnant women with a gestational age of 38‒40 weeks were 1.9 times (AOR= 1.9, 95% CI= 1.0– 3.4, p=0.04) were more likely to be colonized by potential neonatal disease-causing bacteria. All E. coli, Klebsiella species, and Acinetobacter species were susceptible to gentamicin and imipenem. All S. aureus were susceptible to penicillin, tetracycline, clindamycin, and erythromycin.Conclusion: High proportion of pregnant women in this study were colonized with potential neonatal disease-causing bacteria. E. coli was the predominant bacteria. Most bacteria isolated in this study were susceptible to antimicrobial agents tested. Gestational age was significantly associated with the colonization rate of potential neonatal disease-causing bacteria.Keywords: vaginal colonization, pregnant women, neonatal disease, antibiotic susceptibility, Hawassa, Ethiopia