This study created an experimental model to assess the effects of early-life exposures to antibiotics on the intestinal microbiota and gene expression in the brain. For this study, C57BL/6 mice were breed for 5 days and the pregnant dams were randomized into 3 treatment groups. First group was treated with low-dose penicillin G in their drinking water starting during the last week of pregnancy and continuing to postnatal day 10 (PND10). Second group was treated with low-dose penicillin G in their drinking water starting at birth and continuing to PND10. Third group didn’t receive antibiotics in the drinking water and served as controls. The dataset includes 16S rRNA and RNA sequencing data. The data revealed exposing newborn mice to low-dose penicillin led to notable changes in intestinal microbiota population structure and composition. There were also significant effects on frontal cortex and amygdala gene expression, which affected multiple pathways underlying neurodevelopment.