Objective To explore the effect of antibiotic application timings on maternal and infant outcome, bacterial distribution in fetal membranes, and serum C⁃reactive protein (CRP) and procalcitonin levels in patients with preterm premature rupture of membranes (PPROM). Methods The clinical data of 90 PPROM patients were retrospectively analyzed. Patients were assigned to groups A, B, and C according to the application timings of antibiotics, with 30 cases in each group, therein groups A, B, and C received administration of antibiotics within 12-24 hours, 24-36 hours, and 36-48 hours after premature rupture of membranes. The maternal and infant outcome, bacterial distribution in fetal membranes, and levels of serum CRP and procalcitonin were compared between patients of the 3 groups. Results There was no statistically significant difference in the incidence rates of chorioamnionitis, postpartum hemorrhage, and puerperal infection between the 3 groups (P>0.05). There were statistically significant differences in 10⁃minute post⁃delivery Apgar score, and in the incidence rates of neonatal pulmonary infection, intrauterine distress, neonatal asphyxia, and neonatal sepsis in early onset type between the 3 groups (P<0.05), therein group A exhibited higher 10⁃minute post⁃delivery Apgar score, and lower incidence rates of neonatal pulmonary infection, intrauterine distress, and neonatal sepsis in early onset type as compared with group C; moreover, groups A and B yielded a lower incidence rate of neonatal asphyxia as compared with group C (P<0.05). There were statistically significant differences in positive rate of bacterial culture in fetal membranes and mixed bacteria growth rate between the 3 groups (P<0.05), therein group A demonstrated a lower positive rate of bacterial culture in fetal membranes as compared with groups B and C, whereas group C expressed a higher growth rate of mixed bacteria as compared with groups A (P<0.05). After 24-36 hours of delivery, there were statistically significant differences in levels of serum CRP and procalcitonin between the 3 groups, and which were higher than those when patients were admitted (P<0.05), therein serum CRP level was in an ascending order as follows: group A, group B, and group C, and serum procalcitonin level of groups A and B was lower than that of group C (P<0.05). Conclusion For PPROM patients, early prophylactic use of antibiotics can ameliorate neonatal outcome, reduce the incidence rate of neonatal asphyxia and neonatal infection rate, and decrease the levels of serum CRP and procalcitonin.