Preterm birth is defined as delivery before 37 completed weeks of pregnancy, and it is the leading cause of neonatal morbidity and mortality. Oxidative stress is recognized as an important factor in the pathogenesis of premature labor. We conducted this analysis to investigate the safety of administration of the tocolytic drug Atosiban-a reversible, competitive antagonist of the oxytocin receptor in the treatment of preterm birth and its impact on the level of oxidative stress in pregnant women after 48 hours of tocolytic treatment. This prospective study was conducted between March 2016 and August 2017 at the Obstetric Clinic of the Polish Mother's Memorial Hospital Research Institute. Total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI) values as well as 3-nitrotyrosine, carbonyl, and thiol group levels were measured using an ELISA test in serum and plasma of 56 pregnant women before and after 48 hours of continuous administration of Atosiban. We found that TAS levels decreased almost twice after the 48-hour drug administration (0.936 ± 0.360 mmol/L vs. 0.582 ± 0.305 mmol/L, P < 0.001) while TOS increased from 18.217 ± 16.093 μmol/L to 30.442 ± 30.578 μmol/L (P < 0.001). We also found a significant increase in OSI index-almost a threefold increase from 0.022 ± 0.022 to 0.075 ± 0.085, P < 0.001. In addition, statistically significant differences in the level of carbonyl groups were found. It increased from 65.358 ± 31.332 μmol/L to 97.982 ± 38.047 μmol/L (P < 0.001), which indicates increased oxidation of plasma proteins. Furthermore, patients who gave birth prematurely had higher levels of TOS after a 48-hour drug administration than the second group with labor after 37 weeks of pregnancy (42.803 ± 34.683 μmol/L vs. 25.792 ± 27.821 μmol/L, P < 0.031). The obtained results clearly indicate that pregnant women during tocolytic treatment with Atosiban are in a state of increased oxidative stress and occurrence of preterm birth can be associated with this phenomenon. This trial is registered with NCT03570294.