<p>Keystone species and ecosystem engineers play critical roles in maintaining and enhancing biodiversity by modifying their environment and creating ecological niches that support a wide range of organisms. The gopher tortoise (<i>Gopherus polyphemus</i>) is a prime example in the longleaf pine sandhill ecosystems of southeastern United States. Although the gopher tortoise’s positive influence on aboveground biodiversity (especially vertebrates and plants) is well documented, its impact on belowground biodiversity remains largely unexplored. Additionally, it is unclear whether these effects are consistent across landscapes of varying environmental quality (e.g., intact vs. anthropogenically altered ecosystems). To address these gaps, we conducted an observational study in Florida, focusing on two primary microhabitats associated with gopher tortoise activity: burrowing sites vs. foraging areas. These areas were examined within two distinct vegetation types: intact sandhills and degraded pastures. Soil samples were collected and analyzed using molecular methods to characterize belowground communities, including nematodes and microbes, as well as soil biogeochemical properties. We observed that gopher tortoise activities significantly influenced belowground communities. In burrowing areas, microbial (bacteria, fungi, and protists) and bacterial-feeding nematode diversity was generally lower compared to foraging areas. Vegetation type also played a key role, with degraded pastures supporting lower diversity of bacteria, fungi, and protists. Moreover, the magnitude of gopher tortoise effects varied by vegetation type. In sandhill, gopher tortoise activity had a strong effect on microbial diversity whereas the impact was more pronounced for nematode diversity in pasture. These results indicated that the effects of gopher tortoises on nematodes are indirect and are a result of trophic interactions between nematodes and microbial communities. These shifts in soil biodiversity were closely linked to changes in soil biogeochemistry, especially pH and C dynamics, driven by gopher tortoise behavior. From a conservation perspective, our results highlight the significant role of gopher tortoises in shaping soil biodiversity, but their role is constrained by anthropogenic influence on the plant community. Although they retain some capacity to create new niches in pasture, their ability to fully restore all components of soil biodiversity appears limited by the historical anthropogenic influence on the vegetation type. The distinct microbial and nematode communities observed across vegetation types suggest that some structural and functional shifts in soil ecosystems are not recovered by gopher tortoise ecosystem engineering.</p>