Background Prenatal glucocorticoids are commonly used in pregnancies before 34 weeks of gestation in women at risk of preterm delivery; however, the effects of these drugs on late preterm infants (those born after the 34th week of pregnancy) are controversial. As a result, we aimed to investigate the effect of a single course of betamethasone (two doses of betamethasone every 24 hours) on the neonatal outcome of late preterm births. Methods We retrospectively assessed all spontaneous late preterm births (34-36+6 weeks of gestation) at a tertiary hospital in Iran over a period of two and a half years. Exclusion criteria included multiple pregnancies, induced labor, and fetal malformations identified in sonograms. Neonatal outcome measures encompassed first and five-minute Apgar scores, respiratory distress syndrome, neonatal death, birth asphyxia, and the need for positive pressure ventilation, continuous positive airway pressure, tracheal intubation, and surfactant. Baseline characteristics, such as maternal age, parity, fetal gender, and high-risk pregnancy, were considered confounding variables. High-risk pregnancies were defined as any cases involving prolonged rupture of membranes or maternal comorbidities such as severe anemia, preeclampsia, diabetes, or COVID-19. Results During the study period, there were 830 spontaneous preterm births at our center. Of these, only 195 (23.5%) received complete doses of betamethasone. Low birth weight was more common in mothers who did not receive betamethasone compared to those who did (63.6% vs. 41.2%). The mean gestational age was lower in mothers who received betamethasone than in those who did not. Respiratory distress syndrome was more common in mothers who received betamethasone (P<0.001, RR 2.11, 95% CI (0.98-4.18)). However, after adjusting for confounding factors, such as gestational age and birth weight, betamethasone did not increase the risk of respiratory distress syndrome. Other adverse neonatal outcomes did not differ significantly. Conclusions There were no differences in neonatal outcomes between those who received betamethasone and those who did not.