We aimed to determine the relationship of douching prior to pregnancy and bacterial vaginosis (BV) during pregnancy on preterm birth, addressing individual and joint effects. We used a prospective cohort study and assessed vaginal microflora using gram stains and Nugent's criteria. Douching behaviour was based on self-report about the 12 months prior to pregnancy. Preterm births were categorised as spontaneous or medically indicated. A total of 2561 women provided vaginal specimens and 1492 provided self-reports on douching behaviour. Bacterial vaginosis assessed at 24-28 weeks' gestation in the absence of douching prior to pregnancy was associated with spontaneous preterm birth (odds ratio = 2.74 [95% confidence interval 1.13, 6.66]) as was douching in the absence of BV (OR = 2.20 [1.29, 3.75]). The combination of BV and douching was unrelated to spontaneous or medically indicated preterm birth. We concluded that acute alterations in vaginal microflora at mid-pregnancy or douching prior to pregnancy were associated with an increased risk of preterm birth, but the combination did not appear to increase the risk further than would be expected.