PurposeBacterial vaginosis (BV) is quite common and linked with serious public health issues such as premature delivery and spread of sexually transmitted infections. The study aims to identify different genital mycoplasmas (GM) in high vaginal swabs (HVS) from adult females in Bahrain.Design/methodology/approachIn total, 401 HVS were collected and cultured on MYCOFAST® RevolutioN 2 test for identification and antibiotic susceptibility. Polymerase chain reaction (PCR) was performed for detection of Mycoplasma genitalium (Mg), Mycoplasma hominis (Mh) and Ureaplasma species. DNA-probe based detection for Gardnerella, Candida and Trichomonas was performed by BD Affirm Assay. Representative PCR amplicons were sequenced by Sanger sequencing.FindingsIn PCR, Ureaplasma sp. was the most common GM, followed by Mg and Mh; the prevalence being 21.2, 5.2 and 1.5%, respectively. On the contrary, 10.7% samples showed positivity for Ureaplasma urealyticum (Uu) and 1.7% for Mh in MYCOFAST® RevolutioN 2. The concordance rates between MYCOFAST® RevolutioN 2 and PCR for Mh and Ureaplasma sp. were 97.7 and 84%, respectively. Considering PCR as gold standard, sensitivity, specificity, positive predictive value, and negative predictive value of MYCOFAST® RevolutioN 2 were 33.3, 98.8, 28.6, 98.9 and 37.7, 96.5, 74.4, 85.2% for Mh and Ureaplasma sp., respectively. The Uu and Mh isolates showed antibiotic-resistance ranging from 53%–58% and 71%–86%, respectively.Research limitations/implicationsThe prevalence of Ureaplasma sp. was high. Significant co-occurrence of GM was noticed with BV. MYCOFAST® RevolutioN 2 had lower detection-rate than PCR, so a combination is suggested for wider diagnostic coverage.Practical implicationsThe research reflects on status of prevalence of GM in adult females in Bahrain, and their co-occurrence with bacterial vaginosis. Diagnostic approach with combination of tests is suggested for wider coverage. The research has epidemiologic, diagnostic, and therapeutic implications.Originality/valueThis is the first report from the Kingdom of Bahrain reflecting on burden of GM from this geographic location. The diagnostic efficacy of MYCOFAST® RevolutionN 2 test and polymerase chain reaction was evaluated for GM detection.