Aberrant microbial colonization of premature infants is increasingly recognized as a risk factor for severe acute morbidities. The aim of this study was to evaluate the correlation of bacterial upper airway colonization within the first 6 weeks of life in preterm infants <1000g and risk of moderate/severe bronchopulmonary dysplasia (BPD). In this retrospective two-center cohort study postnatal upper airway bacterial colonization of premature infants with a birth weight <1000g was analyzed. Bacteria were categorized into facultative- and highly pathogenic. Within 242 infants, a birth weight cutoff of 800g prevailed as the most relevant discriminator for risk of BPD. Furthermore, center, male sex, duration of antibiotic therapy, and delayed detection of facultative pathogenic bacteria after week 4 was associated with the development of BPD. Using classification tree analyses for the binary outcome, antibiotic therapy was more importance in infants <800g, whereas in those with a birth weight ≥800g, delayed colonization with facultative pathogenic bacteria was more relevant than antibiotic exposure. We add delayed colonization of the upper airway with facultative pathogenic bacteria to the risks for BPD. The variations of microbial colonization should be considered in future studies on the pathogenesis of BPD and new treatment modalities.