In 1989, we observed in our neonatal intensive care unit (NICU), an increased number of infants with gastrointestinal signs, including five cases of necrotizing enterocolitis. Clostridium perfringens was found in 26% of newborns (n = 168) and was associated significantly with the occurrence of flatulence, distended abdomen, foul-smelling stools, diarrhea and blood in stool (all p < 0.001). C. difficile was found in 17% of the newborns (n = 72). Cesarean section, low gestational age and low birth weight were significantly associated with C. perfringens in stools (all p < 0.001). Treatment with antibiotics was not associated with occurrence of C. perfringens. However, in infants with C. perfringens, intrapartum antibiotics were associated with increased appearance of abdominal distension (p < 0.05). Thus the antibiotics, which disturb primary colonization, may also favor the pathogenic role of opportunistic gut bacteria, such as C. perfringens.