During pregnancy, colonization by genital mycoplasmas may be associated with adverse outcomes. This study was conducted to investigate the prevalence of four species of Mollicutes (Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum) in pregnant women receiving high-risk prenatal care and to evaluate possible associated factors. Data collection included the application of a questionnaire and the collection of cervical swabs from pregnant women. Species identification was performed by real-time PCR. The overall prevalence of Mollicutes was 60.97%. 55.9% of pregnant women were colonized by Ureaplasma spp., and 19.51% by Mycoplasma spp. The prevalence rates by species were 48.78% for U. parvum, 11.59% for U. urealyticum, 18.9% for M. hominis, and 1.22% for M. genitalium. Age, 12 years of schooling or more, age at first sexual intercourse up to 14 years, third trimester of pregnancy, having undergone infertility treatment, presence of STI, and groin lymph nodes were associated with a higher prevalence of microorganisms. The results presented are of utmost importance for understanding the prevalence of these microorganisms, the characteristics of colonized pregnant women, and planning screening strategies and interventions that minimize the negative impacts of these infections.