Fungi and bacteria co-inhabit a wide variety of habitats, and their interactions are significant drivers of many ecosystem services and functions. Creating unique microenvironments, fungal mycelia and their surroundings (defined here as the mycosphere) allow for spatially distinct fungal bacterial activities and interactions at the microscale. Fungi in particular modulate the mycosphere pH and oxygen as the drivers and/or the results of various fungal processes. However, due to the microscopic diameters of hyphae (typically 2-10 μm), it is experimentally difficult to non-invasively access themycosphere to thereby analyze the local pH and oxygen on hyphae or around mycelia. Hence, in this thesis, I aimed to develop and deploy microscale techniques to analyzethe mycosphere pH and oxygen in vitroand thereby to further resolve their influences on the local microbial life for a better understanding of mycosphere habitat properties and functioning.