This study investigates the taxonomic composition, richness and diversity of microbial and metazoan communities in two adjacent ecosystems — a preserved Native Forest and a Highland Grassland — within a well-conserved area of the Atlantic Forest located in the southernmost state of Brazil. Topsoil was analysed using high-throughput sequencing and metabarcoding to assess bacterial, archaeal and eukaryotic communities, divided into microeukaryotes and metazoans, therefore representing four groups of organisms. Bacterial communities were largely composed of Pseudomonadota, Acidobacteriota and Actinomycetota, while archaeal communities were dominated by Thermoproteota. Unicellular eukaryotes showed habitat-specific dominance patterns, with Cercozoa prevailing in Native Forest and Apicomplexa in Highland Grassland; metazoans were represented mainly by Annelida and Nematoda. Alpha diversity analysis revealed differences in richness and in two diversity indices (Shannon and Simpson), which were significantly higher in the Native Forest, with the ordination analyses showing clear spatial structure. Zeta diversity analyses revealed taxon-specific turnover dynamics and indicated that drivers of community structure varied across organism categories. Co-occurrence networks amongst eukaryotes showed well-structured associations within habitats. These results underscore the ecological complexity of even closely-situated habitats and emphasise the importance of long-term conservation of these environments. They also highlight the value of multi-site biodiversity frameworks, such as zeta diversity in capturing fine-scale community differentiation across ecosystems.