This chapter is a revision of the realm of endophytic fungi, focusing on how to approach the study of their biodiversity in relation to distribution, richness, host plant defense mechanisms, chemistry, and metabolomic profiles. We will revise the current isolation and identification methods available such as culture-dependent techniques and omics approaches including both targeted and functional metagenomics, and their comparison to give a holistic view of the endophytic fungal biome. Additionally, we will discuss their biotechnological potential for the production of bioactive natural products to be applied in plant protection (against nematodes, fungi, and insect control), soil restoration, or disease control practices based on microbial antagonists.