In order to evaluate factors influencing fetal fibronectin concentrations during pregnancy a total of 312 vaginal samples for fetal fibronectin measurements and microbiological culture were collected and later included in a stepwise regression analysis. Preterm premature rupture of membranes, a modified Bishop score ≥6 (p < 0.0001), a low vaginal polymorphonuclear leukocyte count (p < 0.002), the presence of Enterococcus faecalis (p < 0.0001), Prevotella species (p < 0.05), a bacterial vaginosis-associated flora (p < 0.05), and a non-lactobacilli-dominated vaginal flora (p < 0.05) were associated with an elevated vaginal fetal fibronectin concentration.