Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late-onset sepsis in neonates. The question is whether neonates acquire endemic hospital-adapted clones or incidentally occurring CoNS strains after birth during their hospital stay. Therefore, a prospective study was performed on the prevalence of CoNS in the stool of babies (born vaginally or by cesarean section) during their first days of life. Their clonal relatedness and potential to induce invasive disease were characterized. CoNS were analyzed from the stool samples of newborns with a load of CoNS above 103 colony-forming units (CFU)/mL. The identification of CoNS was performed phenotypically and genotypically. For typing, repetitive polymerase chain reaction (PCR), pulsed-field gel electrophoresis, and multilocus sequence typing were used. Resistance profiles, biofilm production, the presence of icaAD and of IS256 were determined as well. From a total of 207 stool samples (56 newborns), CoNS were detected in 41% of the newborns, mostly on day 3 for the first time (62.5%). Staphylococcus epidermidis was isolated in 85.7% of cases, harbored no IS256 element, and mostly expressed no biofilm. The isolates were separated into four main clusters by repetitive sequence-based PCR. 24% of the strains showed no antimicrobial resistance. 20% were resistant against four antibiotics of two different antibiotic classes. The remaining strains were resistant only against one antimicrobial substance class. Thus, it can be concluded that newborns do not acquire hospital-adapted endemic, multidrug-resistant S. epidermidis isolates during their first days of life. Yet, the results support the thesis that, during hospital stay, environmental parameters may convert sensible/noninvasive S. epidermidis strains into multidrug-resistant strains with characteristics of invasiveness.