The ability to define and characterize the variety of unicellular yeasts and filamentous fungi that inhabit the human gastrointestinal (GI) tract is hampered by the lack of suitable methodologies. This chapter describes culture-based protocols to isolate and culture viable enteric fungi from fresh or refrigerated human fecal samples. It also describes non-culture-based protocols to isolate fungal DNA from human fecal samples to use as a template for fungal community profiling using a high-throughput internal transcribed spacer region 1 (ITS1) sequencing approach. By addressing the major challenges specific to ITS datasets and presenting methods to circumvent them, researchers can select appropriate approaches to ensure robust and meaningful ITS data interpretations.