Enterococci are among the first lactic acid bacteria to colonize the neonatal gastrointestinal tract, but they are also characterized as significant nosocomial pathogens. The aim of this study was to investigate the incidence of antibiotic resistance in enterococci isolated from neonates' gut microbiota as well as the presence of genetic determinants encoding for certain antibiotic resistance traits. A total of 263 fecal samples derived from 97 infants were collected on day 4, 30, and 90 after delivery. Enterococcus faecalis was the most frequently identified species (54.6%) followed by E. faecium, while E. casseliflavus/E. flavescens and E. gallinarum were also traced. The isolates were examined for their resistance to 12 antibiotics. Rifampicin resistance was the highest observed (53.2%), followed by resistance to tetracycline (42.0%), erythromycin (35.7%), and vancomycin (11.2%). Multiresistant strains were highly prevalent. Only intrinsic vancomycin resistance (vanC1 and vanC2/C3) was traced. The ermB gene was detected in 49 out of 96 erythromycin-resistant isolates, while tet genes were detected in 51 out of 113 tetracycline-resistant strains, with tet(L) being the most frequently observed. In conclusion, antibiotic-resistant enterococci are already established in the fecal microbiota of healthy neonates, from the first days of an infant's life.