We usually refer to the critical period for intestinal flora establishment as infancy because the infant gut microbiota is characterized by low diversity and poor stability compared with that of adults. Moreover, it is also vulnerable to interference from a variety of factors. As β-lactam antibiotics are typically used in newborn infants with infectious diseases, we used 16S rDNA sequencing and LC-MS metabolomics to analyze fecal microbes and metabolites in 16 late preterm infants with or without β-lactam antibiotic treatment. The subjects were assigned to two groups: one not treated with antibiotics and another receiving β-lactam antibiotic treatment for less than seven days. Significant changes in fecal microbes and metabolites were observed in the late preterm infants treated with antibiotics, including a reduction in the diversity of the gut microbiota overall and some beneficial bacteria such as Bacteroides, whereas some opportunistic pathogenic bacteria such as Enterococcus showed an overgrowth trend. In addition, significant changes in some crucial metabolites were observed, such as amino acids and bile acids. These findings show that treatment with β-lactam antibiotics might affect the intestinal flora and its metabolites in late preterm infants in a short time period.