Antibiotics, as the first choice for treatment of infections, have been widely used in neonatal units. The application of antibiotics during neonatal period eliminated invasive infections, and improved clinical outcomes. Over the past decades, the effects of antibiotics on the establishment of intestinal microbiota have been recognized. The alteration of intestinal microbiota leads to numerous adverse consequences, which brings challenges to clinical decisions on antibiotic use. We reviewed the effects of the exposure of antibiotics on gut microbiota and succeeding outcomes in neonates to provide new considerations for clinical practices.