This study investigates the response of soil microbial community to spontaneous afforestation - a natural rewilding process that has been ongoing for decades across both national and European levels following land abandonment. The main objectives are to: i) characterize changes in topsoil physico-chemical properties and the associated microbial community response along the successional gradient, ii) assess causal relationships between soil variables and microbial diversity and composition. The research was conducted in two Italian sites: the Foreste Casentinesi National Park (CF) and Julian Prealps (JP). In both areas, five successional stages were identified based on historical orthophotos (1954-2020) and replicated across four chronosequences: meadow-pasture (G), early (E), early-mid (E-M), mid-late (M-L), and late (L) stages of afforestation. Topsoil samples (0–10 cm depth) were analysed for pH, bulk density (BD), organic carbon (OC) and total nitrogen (N). Soil microbial communities were characterized through environmental DNA extracted from fine soil fractions, followed by DNA metabarcoding using ITS and 16S rRNA gene markers for fungi and bacteria, respectively. The results reveal that, along the afforestation gradient, topsoil becomes increasingly acidic and less compact (lower BD), with a concurrent rise in organic matter content. However, these trends are modulated by site-specific variability. Notably, overall microbial diversity was lower at CF compared to JP, a pattern likely linked to more homogeneous environmental conditions and reduced above-ground diversity. Bacteria and fungi exhibited distinct responses to forest regrowth following meadow abandonment. Fungal communities - mainly composed of Ascomycota and Basidiomycota – peaked in diversity at intermediate successional stages. In contrast, bacterial communities, dominated by Proteobacteria and Verrucomicrobiota, showed greater site specificity, especially at lower taxonomic rank. Overall, the findings highlight the ecological relevance of nature-based solutions such as rewilding, not only in fostering microbial diversity but also in contributing to climate neutrality and biodiversity conservation at larger scales.