Sepsis is one of the most important complications in preterm infants. For this reason, most preterm infants receive antibiotics during their first postnatal week. Since 2013, a weekly colonization screening has been installed in German neonatal intensive care units (NICUs), including multi-drug resistant organisms (MDRO) and pathogens with increased epidemic potential. We here investigated the impact of early antibiotic exposure on the colonization with these pathogens. Data from 1407 preterm infants with gestational age < 32 + 0 weeks and born in three NICUs in Germany between January 2014 and December 2019 were analysed. Antibiotics were administered to 911/1407 (64.7%) participating infants during their first postnatal week. Screening-targeted pathogens were detected in 547/1407 (38.9%). Early antibiotic exposure did not increase the risk of colonization with screening-targeted pathogens. The only independent risk factor for colonisation with potential pathogens was the admitting hospital. Interestingly, longer antibiotic therapy (> 7 days) decreased the risk for acquiring pathogens with increased epidemic potential. Early antibiotic exposure did not impact the risk for colonization with MDRO or highly epidemic pathogens in preterm infants. Further studies are needed to identify risk factors for the acquisition of MDRO and highly epidemic pathogens and potential associations with long-term outcome.