Plants interact with a broad range of microorganisms, such as bacteria and fungi. In plant roots, complex microbial communities participate in plant nutrition and development as well as in the protection against stresses. The establishment of the root microbiota is a dynamic process in space and time regulated by abiotic (e.g., edaphic, climate, etc.) and biotic factors (e.g., host genotype, root exudates, etc.). In the last 20 years, the development of metabarcoding surveys, based on high-throughput next-generation sequencing methods, identified the main drivers of microbial community structuration. However, identification of plant-associated microbes by sequencing should be complemented by imaging techniques to provide information on the micrometric spatial organization and its impact on plant-fungal and fungal-fungal interactions. Laser scanning confocal microscopy can provide both types of information and is now used to investigate communities of endophytic, endomycorrhizal, and ectomycorrhizal fungi. In this chapter, we present a protocol enabling the detection of fungal individuals and communities associated to the plant root system.