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Objective To compare the frequency of Ureaplasma-positive gastric fluid (GF) cultures based on the cause and mode of delivery in preterm newborns. Design Retrospective cohort study. Setting A single university hospital in South Korea. Population Women with a singleton pregnancy who delivered prematurely (between 23+0 and 32+0 weeks of gestation, N=464). Methods The newborns’ GF was obtained on the day of birth via nasogastric intubation. The frequency of Ureaplasma-positive GF cultures was compared according to the cause and mode of delivery. Main outcome measures Ureaplasma spp.-positivity in GF cultures. Results Ureaplasma spp. was detected in 20.3% of the GF samples. The presence of Ureaplasma spp. was significantly higher in the spontaneous preterm birth group than in the indicated preterm birth group (30.2% vs 3.0%; P < 0.001). Additionally, Ureaplama spp. was more frequently found in the vaginal delivery group than in the cesarean delivery group, irrespective of the cause of preterm delivery [indicated preterm birth group (22.2% vs. 1.9%, P = 0.023); spontaneous preterm birth group (37.7% vs. 24.2%, P = 0.015)]. Conclusions Ureaplasma spp. were found in 20.3% of the GFs. However, only 1.9% of newborns in the indicated preterm birth group with cesarean delivery had a Ureaplasma-positive GF culture. Our findings support the view that the amniotic cavity is a sterile environment before the onset of parturition. Funding Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea (Grant No. HI22C1859) and the Seoul National University Bundang Hospital Research Fund (Grant No. 14-2021-0025).