Despite widespread use of antibiotics, few studies have measured their effects on the burden or diversity of bacteria in the mammalian intestine. We developed an oral antibiotic treatment protocol and characterized its effects on murine intestinal bacterial communities and immune cell homeostasis. Antibiotic administration resulted in a 10-fold reduction in the amount of intestinal bacteria present and sequencing of 16S rDNA segments revealed significant temporal and spatial effects on luminal and mucosal-associated communities including reductions in luminal Firmicutes and mucosal-associated Lactobacillus species, and persistence of bacteria belonging to the Bacteroidetes and Proteobacteria phyla. Concurrently, antibiotic administration resulted in reduced RELM beta production, and reduced production of interferon-gamma and interleukin-17A by mucosal CD4(+) T lymphocytes. This comprehensive temporal and spatial metagenomic analyses will provide a resource and framework to test the influence of bacterial communities in murine models of human disease.