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© 2016 The Author(s). Background: Detection of Ureaplasma, Mycoplasma and Candida spp. in the vagina during pregnancy has previously been associated with preterm birth (PTB). However, the prevalence of these microorganisms and the associated obstetric risks (likely to be population-specific) have not been determined in Australian women; furthermore, in the case of Ureaplasma spp., very few studies have attempted characterisation at the species level and none have examined genotype/serovar status to further refine risk assessment. Methods: In order to address these issues we sampled the vaginal fluid of 191 pregnant Australian women at three time points in pregnancy. Culture methods were used for detection of Ureaplasma spp. and Candida spp., and real-time PCR was used for speciation of U. parvum and U. urealyticum, non-albicans Candida spp., Mycoplasma hominis and Mycoplasma genitalium. High-resolution melt PCR was used to genotype U. parvum. Data on various lifestyle factors (including sex during pregnancy and smoking), antimicrobial use and pregnancy outcome were collected on all participants. Chi-square tests were used to assess the association of vaginal microorganisms with PTB. Results: Detection of Ureaplasma spp. was higher among spontaneous PTB cases, specifically in the presence of U. parvum [77 % preterm (95 % confidence interval (CI) 50–100 %) vs. 36 % term (CI: 29–43 %), p = 0.004], but not U. urealyticum. The association with PTB strengthened when U. parvum genotype SV6 was detected (54 % preterm (CI: 22–85 %) vs. 15 % term (CI: 10–20 %), p = 0.002); this genotype was also present in 80 % (4/5) of cases of PTB <34 weeks gestation. When present with Candida albicans in the same sample, the association with PTB remained strong for both U. parvum [46 % preterm (CI: 15–78 %) vs. 13 % term (CI: 8–18 %), p = 0.005] and U. parvum genotype SV6 [39 % preterm (CI: 8–69 %) vs. 7 % term (CI: 3–11 %), p = 0.003]. With the exception of Candida glabrata, vaginal colonisation status for all organisms was stable throughout pregnancy. Smoking significantly increased the likelihood of detection of all target organisms. Conclusions: These data suggest that the presence of different species and serovars of Ureaplasma spp. in the vagina confers an increased risk of spontaneous PTB, findings which may be useful in risk assessment for identifying women who would benefit from antimicrobial treatment.