Objective To analyze the colonization and influencing factors of oral microbiota in preterm infants at different time points of mechanical ventilation, and provide a basis for the development of targeted intervention measures. Methods Clinical data of 97 premature infants from the neonatal intensive care unit (NICU) in a tertiary first-class maternal and child health care hospital from July 2021 to December 2022 were selected. Oral microbiota of premature infants 0, 12, 24, 48 and 72 hours after mechanical ventilation were detected. Correlation and the influencing factors between oral microbiota colonization and duration of mechanical ventilation were analyzed. Results A total of 485 specimens from premature infants were collected for oral microbiota detection, out of which 127 specimens (26.19%) were positive. Detection rates of oral microbiota at different time points were positively correlated with duration of mechanical ventilation (r=0.292, P < 0.01). Pairwise comparison showed statistically significant differences in the detection rates of oral microbiota between 0 hour and 24, 48, 72 hours, as well as those between 12 hours and 48, 72 hours, respectively (all P < 0.05). Among 97 premature infants, 65 cases had oral microbiota colo- nization, with a colonization rate of 67.01%; 146 strains were detected, including 94 Gram-positive bacterial strains (64.38%) and 52 Gram-negative bacterial strains (35.62%). The top 5 pathogenic bacteria were Streptococcus oralis, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae, and Staphylococcus hemolyticus. Univariate analysis showed that gestational age at birth, birth weight, lactation within 72 hours, and the use of antimicrobial agents were associated with oral microbiota colonization. Logistic regression analysis showed that birth weight ≥1 500 g (OR=0.102, 95%CI: 0.017-0.634) and lactation within 72 hours (OR=0.290, 95%CI: 0.107-0.783) were independent protective factors for oral microbiota colonization (both P < 0.05). Conclusion Oral microbiota of premature infants underwent mechanical ventilation should be detected as soon as possible within 24 hours after treatment, and targeted intervention measures should be carried out based on changes in microbiota. Breastfeeding or oral care with breast milk should be given as soon as possible. If breast milk is inaccessible, donated human milk or premature infant formula milk should be adopted within 72 hours.