Objective: The objective of this study is to assess the relationship between non-invasive ventilation (NIV) and the colonisation of oral and nasal microbiota in preterm infants within the neonatal intensive care unit (NICU).Design: A prospective cohort study.Setting: The NICU of Zhejiang University Children’s Hospital.Patients: Patients include preterm infants with a gestational age of 28–35 weeks, enrolled within the first 24 hours of life.Interventions: Infants were categorised based on respiratory support: NIV, which included nasal continuous positive airway pressure, nasal intermittent positive pressure ventilation or high-flow nasal cannula; and no respiratory support, defined as room air or low-flow nasal cannula at ≤2 L/min.Main outcome measures: The primary outcome was the colonisation of oral and nasal microbiota at 5 days post birth, measured by colony-forming units per millilitre (CFU/mL), with colonisation defined as bacterial growth >10³ CFU/mL.Results: The study included 100 preterm infants, with 50 in each group. Nasal microbial colonisation was observed in 56% (28/50) of the NIV group, significantly higher than the 28% in the no respiratory support group. No significant differences were found in oral colonisation. Adjusted binary logistic regression showed an association between NIV and increased risk of nasal colonisation (adjusted OR=2.91, 95% CI 1.12 to 7.58, p=0.028).Conclusions: NIV in preterm infants was linked to a higher risk of nasal microbial colonisation. This finding suggests the need for further research and consideration of infection control strategies in the NICU.