Pedogenesis determines soil physicochemical properties and many biodiversity facets, including belowground microbial bacteria and fungi. At the local scale, top-down predation by microbial protists regulates the soil microbiome, while the microbiome also affects protistan communities. However, it remains unknown how pedogenesis affects protistan communities and the potential protist-microbiome predator-prey relationships. With 435 soil samples representing different stages of pedogenesis ranging in soil age from centuries to millennia, we examined the influence of pedogenesis on the main protistan groups, and the interrelationships between protistan predators and microbial prey biomass. We revealed an enrichment in the diversity of total protists across pedogenesis and increasing richness of phototrophic protists in the medium compared with the early stages of pedogenesis. The richness of predatory protists accumulated throughout pedogenesis, which was more strongly determined by microbial biomass than environmental factors. Predator-prey associations were stronger in the young and the medium soils than in the older soils, likely because prey biomass accumulated in the latter and might be no longer limit predators. Together, our work provides evidence that pedogenesis shapes predatory protists differently than their prey, leading to shifts in predator-prey relationships. This knowledge is critical to better understand how soil food webs develop across soil development which might lead to changes in ecosystem functions.