The human gastrointestinal tract is home to a thriving community of microbes including the fungal 'mycobiota'. Although sequencing methodology has enumerated diverse fungal genera within this niche, discerning persistent symbiotic residents from contaminants and purely environmental transients remains a challenge. Recent advances in culturomics and sequencing employing metagenomics, metatranscriptomics and longitudinal studies have begun to reveal a human symbiont 'core mycobiome' that may contribute to human health and disease. Trans-kingdom interactions between the bacterial microbiota and evolution within the niche have defined C. albicans as a true symbiont, setting a bar for defining other fungi. Additionally, elegant investigations of mammalian antifungal immunity have examined mononuclear phagocytes, neutrophils, antigen-specific recognition by T cells and other mechanisms important for local and systemic effects on the host, providing further evidence supporting gut persistence. In this review we discuss current research aimed at investigating the symbiotic mycobiota and propose four criteria aiding in the differentiation of fungal symbionts from environmental transients.