Amniotic fluid infection with Ureaplasma urealyticum or Mycoplasma hominis can cause chorioamnionitis and preterm birth. The aim of this study was to examine whether vaginal Ureaplasma urealyticum/Mycoplasma hominis colonization is predictive of preterm delivery in patients exhibiting signs of threatened preterm birth or those with asymptomatic short cervix. The present retrospective study, which was performed in a perinatal tertiary center, included patients carrying a singleton pregnancy who were referred to the emergency Ob/Gyn unit because of regular preterm uterine contractions and/or short cervical length (<20 mm) at 22-33 weeks of gestation, and in whom a vaginal U. urealyticum/M. hominis examination (Urea-arginine LYO-2, BioMerieux ® ) was performed. Univariate and multivariate analyses were performed to assess the association between vaginal U. urealyticum or M. hominis and chorioamnionitis or preterm delivery. The median gestational age of the 94 enrolled patients was 29.9 weeks, and 54 (57%) of the patients were vaginal U. urealyticum/M. hominis-positive. The preterm delivery rate in the positive group was higher than in the negative group (53 versus 25%; p = .007). Vaginal U. urealyticum/M. hominis positivity was found to be an independent risk factor for preterm birth at <37 weeks of gestation (adjusted odds ratio = 4.0, 95% confidence interval, 1.1-15.3) in a multivariate analysis adjusted for age, history of preterm delivery and conization, gestational age, cervical length, presence of vaginal bleeding, vaginal fetal fibronectin and serum C-reactive protein at test. U. urealyticum/M. hominis positivity was not associated with delivery at <34 weeks or chorioamnionitis. A positive vaginal U. urealyticum/M. hominis culture is an independent predictive factor for preterm birth in patients with symptomatic threatened preterm labor and/or short cervix.