The oral cavity, skin, vagina, and colon are environments equipped with specialized structures and functions which can support complex ecosystem of microorganisms enabling them to serve as protective barriers, regulate immune responses, and contribute to metabolic processes. Samples from these sites are characterized by low biomass, contamination by human DNA, and bacteria recalcitrant to lysis. Recent advances in DNA extraction methodology and metagenomic and high-throughput sequencing technologies have improved the ability to characterize these complex and diverse microbial communities. In this chapter, sample collection methodologies for the oral cavity, skin, vagina, and biopsies from the colon are provided, including preparation of the collection site, choice of sampling tools, sample handling, transport, and storage to minimize microbial degradation and contamination.