BACKGROUND: Sexually transmitted infections (STIs) are a significant public health concern that can lead to serious outcomes such as infertility, pregnancy complications, and neonatal infections. This study aimed to evaluate the prevalence of STI and their associated risk factors in symptomatic and asymptomatic pregnant women. METHODS: Between July and October 2024, a total of 300 pregnant women in their third trimester, including 113 symptomatic and 187 asymptomatic individuals aged 18 to 45 years, who sought antenatal care at the Gynecology and Obstetrics Clinic of Ankara Etlik City Hospital, were included in the study. The detection of STIs agents in vaginal swab samples was performed using multiplex polymerase chain reaction in the Molecular Diagnosis Laboratory of the Department of Microbiology. RESULTS: The overall prevalence of STIs was 34.3% (103/300), with single and multiple infections accounting for 28.3% and 6.0% of cases, respectively. The most frequently detected pathogens were Ureaplasma parvum/urealiticum (29.0%), Mycoplasma hominis (4.6%), and Chlamydia trachomatis (2.3%). Co-infections were commonly observed between Ureaplasma parvum/urealiticum and Mycoplasma hominis. No significant difference in STI prevalence was observed between the symptomatic (35.4%) and asymptomatic (33.7%) groups. Co-infection with non-STI bacterial agents, such as Gardnerella vaginalis and Streptococcus agalactiae, increased the risk of STIs by 1.96 times (p = 0.006). CONCLUSIONS: This study revealed that STIs occur at similar rates among symptomatic and asymptomatic pregnant women. This finding highlights the critical importance of detecting asymptomatic cases to prevent the spread of silent infections and to safeguard maternal and neonatal health. Ureaplasma parvum/urealiticum were identified as the most common pathogens. Given that co-infections with non-STI bacterial agents significantly increase the risk of STIs, multiplex PCR-based multicenter and prospective studies are essential to refine screening strategies for pregnant women.