Host–microbe interactions have been linked to health and disease states through the use of microbial taxonomic profiling, mostly via 16S ribosomal RNA gene sequencing. However, many mechanistic insights remain elusive, in part because studying the genomes of microbes associated with mammalian tissue is difficult due to the high ratio of host to microbial DNA in such samples. Here we describe a microbial-enrichment method (MEM), which we demonstrate on a wide range of sample types, including saliva, stool, intestinal scrapings, and intestinal mucosal biopsies. MEM enabled high-throughput characterization of microbial metagenomes from human intestinal biopsies by reducing host DNA more than 1,000-fold with minimal microbial community changes (roughly 90% of taxa had no significant differences between MEM-treated and untreated control groups). Shotgun sequencing of MEM-treated human intestinal biopsies enabled characterization of both high- and low-abundance microbial taxa, pathways and genes longitudinally along the gastrointestinal tract. We report the construction of metagenome-assembled genomes directly from human intestinal biopsies for bacteria and archaea at relative abundances as low as 1%. Analysis of metagenome-assembled genomes reveals distinct subpopulation structures between the small and large intestine for some taxa. MEM opens a path for the microbiome field to acquire deeper insights into host–microbe interactions by enabling in-depth characterization of host-tissue-associated microbial communities.