Bacterial vaginosis (BV) and genital mycoplasmas are infections of the reproductive tract that play important roles in maternal and foetal health. Genital mycoplasmas include Mycoplasma genitalium, M. hominis, Ureaplasma parvum and U. urealyticum. Infection may increase a woman’s susceptibility to infection with the human immunodeficiency virus (HIV). Bacterial vaginosis associated bacteria may form biofilms that are responsible for antimicrobial resistance and about 30% of affected women will relapse within three months of treatment. Genital mycoplasmas are prone to develop point mutations, which are responsible for increased antimicrobial resistance. Infections with these bacteria become prominent during pregnancy as infection may lead to infertility and foetal death. The purpose of the study was to determine the association between genital mycoplasmas and BV in pregnant women. Pregnant women attending the antenatal and Maternal and Foetal Unit (MAFU) clinics of a tertiary academic hospital in Pretoria, South Africa were included in the study. Self-collected vaginal swab specimens were obtained from consenting women older than 18 years of age. With the aid of microscopy, the Nugent scoring system was used to diagnose BV. Genital mycoplasmas were cultured on A2 agar and were diagnosed and speciated with a multiplex polymerase chain reaction (mPCR) assay. In addition, genital mycoplasmas were diagnosed and the antimicrobial susceptibility profiles determined with the Mycofast Revolution assay. A quantitative real-time polymerase chain reaction (qPCR) was employed to quantify the BV associated bacteria Atopobium vaginae and Gardnerella vaginalis. The prevalence of BV in this study was found to be 17.7% in 220 recruited pregnant women. Threshold concentrations between 106 to 107 copies/reaction of A. vaginae and G. vaginalis were found to be the best predictors of BV. Genital mycoplasmas were poorly recovered from A2 agar media, which had a contamination rate of 54.9%. An mPCR assay revealed that genital mycoplasmas were prevalent in 2.3% to 71.4% of specimens with U. parvum being the most prevalent species. The resistance of Ureaplasma species to tetracycline and erythromycin was 73% and 80%, respectively. Minor resistance to the fluoroquinolones, levofloxacin and moxifloxacin was recorded. This study found that only the genital mycoplasmas, namely M. hominis and U. parvum, were significantly associated with BV, while M. hominis was also significantly isolated from HIV positive women. This study found that there is an association between BV and genital mycoplasmas. The high prevalence of BV and genital mycoplasmas suggests that current management and/or intervention strategies are insufficient. Bacterial vaginosis associated bacteria can form a polymicrobial biofilm, which confer protection against antimicrobial agents and host immune responses. These biofilms are present on genital sites like the endometrium, which is located close to the amniotic membranes, posing health risks for the pregnancy. Future research must focus on the study of in vitro BV biofilm models and effective treatment strategies to minimise antimicrobial resistance. In the meantime, low-cost point-of-care (POC) tests that can accurately diagnose RTIs are needed to prevent excessive and unnecessary administration of antimicrobial agents and improve maternal and foetal health in the South African health care system.