Fungi are both widespread and essential members of environmental and host-associated microbiomes. Their presence and functional roles have a significant impact on microbiome dynamics and broader ecological processes. Despite this, the biological complexity of fungi is often underestimated. Fungi generate a variety of intricate structures throughout their life cycle and contain numerous organelles, lipid droplets, and other intracellular components. Additionally, filamentous fungi can host bacteria (such as cyanobacteria), mycoviruses, and other fungi and are even capable of internalizing entire microalgal cells within their hyphae. Endohyphal symbionts are microorganisms, such as bacteria, that live inside the hyphae of fungi. Isolation and maintenance of these symbionts require careful handling to ensure that the symbionts are separated from the host fungi and maintained under appropriate conditions. In this book chapter, we describe the various protocols for isolating and maintaining endohyphal symbionts from fungi.