Mycoplasma hominis and Ureaplasma urealyticum are associated with chorioamnionitis, preterm delivery and pelvic inflammatory disease. The aim of this study was to evaluate the possible risk factors of co-colonization by M. hominis in patients already colonized by U. urealyticum and compare demographic parameters, vaginal pH and microbiota of women colonized by U. urealyticum or M. hominis. A total of 452 patients positive for U. urealyticum or M. hominis were analysed, 421 (93.1%) of whom were positive for U. urealyticum and 31 (6.9%) for M. hominis. Patients positive for M. hominis compared to patients positive for U. urealyticum were more frequently colonized by Gardnerella vaginalis (71% vs 18.5%; p 0.0001), less frequently by lactobacilli (16.1% vs 61.5%; p 0.0001), and more frequently had a pH value higher than 4.5 (96.8% vs 57%; p 0.0001), all conditions associated to bacterial vaginosis (BV). Logistic regression analysis showed that only G. vaginalis colonization and pH higher than 4.5 were independently related to M. hominis colonization (respectively p 0.0001 and p 0.016). Thus, in women colonized by U. urealyticum, BV is an independent risk factor for M. hominis co-colonization.