A highly sensitive PCR method developed in our university accurately identifies the presence or absence of intra-uterine (IU) microbes without false positive results. With the inclusion of the results of an accurate assessment of IU microbes, risk factors for the development of moderate/severe bronchopulmonary dysplasia (BPD), a chronic lung disease that affects premature infants who require prolonged oxygen therapy or medical ventilation, were examined in 107 spontaneous preterm neonates. Perinatal risk factors were compared between cases of moderate/severe BPD (N = 49) and mild/non-BPD (N = 58). There were no cases of IU Ureaplasma/Mycoplasma infection alone. IU coinfections defined as mixed infections of Ureaplasma/Mycoplasma and other bacteria (bacteria other than Ureaplasma/Mycoplasma) (54.0 %), histological chorioamnionitis ≥II using Blanc’s classification showing a maternal inflammatory response with neutrophil inflammation extending deeper into the chorion and/or amnion (69.4 %), delivery weeks [26 (22 – 32) weeks], and male sex (65.3 %) in moderate/severe BPD cases significantly differed from those in mild/non-BPD cases [16.7 %, 46.5 %, 28 (23−32) weeks, and 44.8 %, respectively] (p < 0.01, p = 0.01, p < 0.01, and p = 0.03, respectively). IU coinfections [odds ratio (OR) 5.4, 95 % confidence interval (CI) 1.7 – 17.0, p < 0.01], neonatal immaturity (OR 4.8, 95 % CI 1.6 – 14.3, p < 0.01), and male sex (OR 3.3, 95 % CI 1.1 – 9.8, p = 0.03) were identified as independent risk factors for the development of moderate/severe BPD. IU coinfections, such as Ureaplasma/Mycoplasma and other bacteria, were associated with the development of moderate/severe BPD.