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Grassland afforestation poses a threat to biodiversity beyond land‐use conversion. Diversity patterns are shaped by temporal dynamics, particularly, time since afforestation can decline beta diversity and lead to biotic homogenisation. Our study examines the effect of grassland afforestation on soil prokaryotic and fungal beta diversity. We evaluate the contributions of colonisation and extinction processes to beta diversity, as well as the replacement of endemic species by ubiquitous ones. Along a 200 km climatic gradient in Argentina's Pampas region, we analysed grasslands and mature eucalypt plantations at different times since afforestation. Soil samples were collected at each site and analysed using 16S (V3–V4) and ITS2 amplicon sequencing to identify prokaryotic and fungal communities, respectively. The analyses revealed biotic homogenisation at the transition from grassland to newly planted stands, evidenced by a decrease in intratreatment beta diversity. Increasing time since afforestation did not exacerbate this decline. However, our findings indicate that there are different responses between prokaryotes and fungi. The homogenisation of prokaryotes in young stands is due to the low heterogeneity in colonising communities. On the other hand, the decline in fungal beta diversity is likely caused by other mechanisms beyond extinction or replacement. The study highlights the impacts of the afforestation process on the beta diversity of soil microbial communities of grasslands, affecting taxonomic groups in different ways. Although microbial diversity may be partially restored in time in eucalypt plantations, it is important to investigate its underlying mechanisms and the ecological implications for microbial diversity and its spatial distribution. [ABSTRACT FROM AUTHOR]