Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and microbial dispersal. Mycelial networks created by filamentous fungi or other filamentous microorganisms (e.g., Oomycetes) act as 'fungal highways' that can be utilized by bacteria for transport throughout heterogeneous environments, greatly facilitating their mobility and granting them access to regions that may be challenging or impossible to reach on their own (e.g., due to air pockets within the soil). Several devices and experimental protocols have been created to study these fungal highways, including fungal highway columns. The fungal highway column designed by our group can be used for a variety of in situ or in vitro applications, as well as with diverse environmental and host-associated sample types. Herein, we describe the methods for performing experiments with these columns, including designing, printing, sterilizing, and preparing the devices. The options for analyzing data obtained from the use of these devices are also discussed here, and troubleshooting advice regarding potential pitfalls associated with experiments using fungal highway columns is offered. These devices can be used to gain a more comprehensive understanding of the diversity, mechanisms, and dynamics of fungal highway BFIs to provide valuable insights into the structural and functional dynamics within complex environments (e.g., soils) and across diverse habitats in which bacteria and fungi co-exist.