Preterm premature rupture of membranes (pPROM) stands as a primary contributor to preterm deliveries worldwide, closely linked to consequential infectious peripartum complications, including chorioamnionitis and early-onset neonatal sepsis. As a prophylactic measure, individuals following pPROM routinely undergo antibiotic treatment. The aim of this study was to evaluate changes in the vaginal microbial colonization after antibiotic treatment following pPROM. Therefore, we retrospectively assessed the impact of antibiotic treatment on the maternal vaginal microbial colonization in 438 post-pPROM patients delivering before 29 gestational weeks. Vaginal samples were collected for microbiological analysis before and after antibiotic administration and analysed for seventeen pre-defined microbial groups. We observed eradication in eleven microbial groups, including beta-hemolytic streptococci group B and Gardnerella vaginalis. No significant reduction was found for the remaining groups, including Escherichia (E.) coli. Moreover, we found a notable increase in resistant bacteria after antibiotic treatment. In conclusion, broad-spectrum antimicrobial treatment exhibited substantial efficacy in eradicating the majority of pathogens in our cohort. However, certain pathogens, notably E. coli, showed resilience. Given E. coli’s prominent role in infectious peripartum complications, our findings underline the challenges in antibiotic management post-pPROM and the need to establish international guidelines, particularly regarding emerging concerns about antibiotic resistances.