Natural grasslands are important reservoirs of animal and plant biodiversity and provide several ecosystem services, through the action of soil microorganisms. The increased demand for food, energy, and cattle activity has led to the conversion of natural grasslands to cultivated systems. However, the consequences of this conversion for soil microbial diversity and ecosystem functioning are yet to be discovered. Here, we used the 16 S rRNA amplicon sequencing and a large set of soil and environmental variables to understand the possible effects of natural grasslands to cultivated pasture conversion on the soil microbial structure, composition, diversity, and functions. The study areas followed a gradient of increasing soil disturbance intensity, as follows: Natural grassland (NG), Improved-natural grassland (IG), Perennial-cultivated pasture (PP), and Annual-cultivated pasture (AP). Natural grassland conversion to managed and cultivated pastures decreased the abundances of Acidobacteria and Verrucomicrobia, while increased α-, γ-, and δ-Proteobacteria, Gemmatimonadetes, Bacteroidetes, Patescibacteria, and Latescibacteria. The predicted functional profiles have also changed, as functions like ‘cellulolytic and symbionts/parasites’ decreased after natural to cultivated pastures conversion, while ‘nitrogen respiration’, ‘sulfur respiration’, and ‘aromatic compound degradation’ functions increased. Aboveground plant diversity decrease influenced belowground microbial diversity. The main drivers of diversity, composition, and functional potential are associated with soil attributes affected by liming, like aluminum complexation. In conclusion, we have found taxonomic and functional differences between natural and managed grasslands (NG and IG, respectively) and cultivated pastures (PP and AP), with consequences for management strategies and biodiversity conservation priorities.