The mucosal-associated microbiome (MAM) of the gastrointestinal tract plays an essential role in human health. Disturbances in the MAM composition has been associated to the pathogenesis of several gut diseases. However, the functional bases of these disturbances are poorly understood, as the recovery of microbial genomes through metagenomics has been hindered by the high ratio of host to microbial DNA. Although several techniques have been developed that can decrease the ratio of host to microbial DNA in such samples, none have been systematically tested on solid mammalian biopsies to recover microbial genomes. Here, five methods were assessed for their ability to effectively deplete host DNA, maintain microbial community structure, enrich the microbiome, and recover microbial metagenomes-assembled genomes (MAGs) from colonic biopsy samples. Each method was initially tested on mice colonic samples and the most effective approach was subsequently applied to human colonic samples. The best method consisted of lysing host cells with saponin prior to the extraction of microbial DNA (Bruggeling et al., 2021). This method reduced the amount of host DNA by up to 14% in human biopsy sample and enabled the detection of 68±99 bacterial species, when only 1.5±1.5 bacterial species can be detected in human biopsy samples at similar sequencing depth without employing a host depletion method (Wu-Woods et al., 2023). Moreover, this approach allowed for the recovery of up to 6 bacterial medium quality MAGs (≥70% completeness and <10% contamination) from some of the most abundant bacteria in human colonic samples. These results provide valuable insights into the efficacy and limitations of host DNA depletion methods for microbiome studies in intestinal samples. Additionally, it highlights the best currently available method that can be employed for the in-depth characterization of mucosal-associated microbiomes to understand their functional role in human health.