Context <p>Land-use change is a major anthropogenic pressure and a leading driver of biodiversity loss globally. However, the responses of soil fungal communities to long-term conservation and restoration interventions remain poorly documented, particularly in African highland landscapes. The Tulu Korma Center for the Restoration of Ethiopia’s Biodiversity and Key Natural Resources provides an opportunity to examine fungal community responses within a long-term restoration landscape in the Ethiopian highlands.</p> Objectives <p>To assess how long-term conservation and restoration interventions, grazing, and cultivated land-use types shape soil fungal diversity, community composition, and functional guild structure within a landscape in central Ethiopia.</p> Methods <p>A stratified random sampling design was used to classify the study landscape into three land-use types: conservation, grazing, and cultivated. Twenty-seven composite soil samples (one per 100 m² plot) were collected across the three land-uses. Fungal DNA was extracted and amplified targeting the Internal Transcribed Spacer 1 (ITS1) region, followed by high-throughput sequencing on the Illumina MiSeq platform. Sequence data were processed and assigned taxonomic and functional identities using the UNITE database.</p> Results <p>A diverse soil fungal community was detected across all land-use types. In total, 428,658 high-quality sequences were clustered into 2750 operational taxonomic units belonging to 15 fungal phyla. The most abundant phyla were Ascomycota (68.79%) and Basidiomycota (24.63%). Fungal richness and relative abundance differed significantly among land-use types (F = 6.456, <i>p</i> = 0.006; F = 5.702, <i>p</i> = 0.009), respectively. The Shannon and Simpson diversity indices did not vary significantly. Several functional guilds, including root endophytes, saprotrophs, and parasites, showed significant differences among land-uses (<i>p</i> &lt; 0.05). Conservation sites supported a diverse assemblage of taxa frequently associated with less disturbed environments (e.g., <i>Penicillium godlewskii</i>, <i>Cystocoleus ebeneus</i>), whereas cultivated lands were dominated by taxa commonly reported from intensively managed or disturbed systems (e.g., <i>Talaromyces stellenboschiensis</i>). Grazing lands harboured distinct taxa such as <i>Cordyceps bassiana</i>. PERMANOVA analysis confirmed significant differences in fungal community composition among land-use types (R² = 0.33, <i>p</i> = 0.001).</p> Conclusions <p>The findings indicate that land-use change is associated with marked shifts in both the taxonomic composition and functional guild structure of soil fungal communities. Conservation areas appear to play an important role in maintaining fungal diversity and functional potential within human-modified landscapes, potentially contributing to the ecological resilience of mixed-use landscapes. These results highlight the importance of considering belowground biodiversity alongside aboveground components when developing land-use planning, restoration, and conservation strategies.</p>