Introduction  Abnormal vaginal flora (AVF) before 14 gestational weeks is a risk factor for preterm birth (PTB). The presence of aerobic microorganisms and an inflammatory response in the vagina may also be important risk factors. Aim  The primary aim of the study was to investigate the differential influences of AVF, full and partial bacterial vaginosis, and aerobic vaginitis in the first trimester on PTB rate. The secondary aim was to elucidate why treatment with metronidazole has not been found to be beneficial in previous studies. Setting  Unselected women with low‐risk pregnancies attending the prenatal unit of the Heilig Hart General Hospital in Tienen, Belgium, were included in the study. Materials and methods  At the first prenatal visit, 1026 women were invited to undergo sampling of the vaginal fluid for wet mount microscopy and culture, of whom 759 were fully evaluable. Abnormal vaginal flora (AVF; disappearance of lactobacilli), bacterial vaginosis (BV), aerobic vaginitis (AV), increased inflammation (more than ten leucocytes per epithelial cell) and vaginal colonisation with Candida (CV) were scored according to standardised definitions. Partial BV was defined as patchy streaks of BV flora or sporadic clue cells mixed with other flora, and full BV as a granular anaerobic‐type flora or more than 20% clue cells. Vaginal fluid was cultured for aerobic bacteria, Mycoplasma hominis and Ureaplasma urealyticum. Outcome was recorded as miscarriage ≤13 weeks + 6 days early miscarriage (EM), n = 8 (1.1%), between 14 + 0 and 24 weeks + 6 days late miscarriage (LM), n = 7 (0.9%), delivery or miscarriage ≤34 weeks + 6 days n = 29 (3.8%), ≤36 weeks + 6 days n = 70 (9.2%). PTB between 25 + 0 and 36 weeks + 6 days was further divided in severe PTB (SPTB, 25 + 0 to 34 weeks + 6 days) and mild PTB (MPTB, 35 + 0 to 36 weeks + 6 days). Results  Women without abnormalities of the vaginal flora in the first trimester had a 75% lower risk of delivery before 35 weeks compared with women with AVF odds ratio (OR) 0.26; 95% confidence interval (CI) 0.12–0.56. The absence of lactobacilli (AVF) was associated with increased risks of PTB (OR 2.4; 95% CI 1.2–4.8), EPTB (OR 6.2; 95% CI 2.7–14) and miscarriage (OR 4.9; 95% CI 1.4–17). BV was associated with increased risks of PTB (OR 2.4; 95% CI 1.1–4.7), EPTB (OR 5.3; 95% CI 2.1–12.9) and miscarriage (OR 6.6; 95% CI 2.1–20.9) and coccoid AV was associated with increased risks of EPTB (OR 3.2; 95% CI 1.2–9.1) and miscarriage (OR 5.2; 95% CI 1.5–17). In women with BV, partial BV had a detrimental effect on the risk of PTB for all gestational ages, but full BV did not. Preterm deliveries later than 24 weeks+ 6 days were more frequent when M. hominis was present (EPTB OR 13.3; 95% CI 3.2–55). Discussion  Bacterial vaginosis, AV and AVF are associated with PTB, especially LM and severe PTB between 25 and 35 weeks. The absence of lactobacilli (AVF), partial BV and M. hominis, but not full BV, were associated with an increased risk of preterm delivery after 24 weeks+ 6 days. As metronidazole effectively treats full BV, but is ineffective against other forms of AVF, the present data may help to explain why its use to prevent PTB has not been successful in most studies.