Preterm neonates are at high risk of infectious and inflammatory diseases which require antibiotic treatment. Antibiotics influence neonatal gut microbiome development, and intestinal dysbiosis has been associated with delayed gastrointestinal transit. Neonates who take less time to pass meconium have a better tolerance to enteral feeding. We analyzed the effect of neonatal antibiotic treatment on the stool pattern and oral tolerance in 106 preterm infants < 33 weeks gestational age. Neonates were classified in 3 groups according to neonatal antibiotic (ABT) treatment days: no antibiotics, 3–7 d ABT, and ≥8 d ABT. Preterm infants from the ≥8 d ABT group took longer to pass meconium and to start green and yellow stools, took longer to reach 100 and 150 mL/kg/day, and reached reduced volumes in enteral feeds at day of life 14 and 28 than infants from no ABT and 3–7 d ABT groups. Multiple linear regression models showed that neonatal antibiotic treatment, birth weight, invasive mechanical ventilation, surfactant, enteral feeding start day, neonatal parenteral nutrition, and neonatal fasting days are associated with the stool pattern and oral tolerance in preterm infants.