The study investigates the long-term effects of organic and conventional farming on soil microbial diversity using a high-throughput pyrosequencing approach. The DOK experiment, which has been running for over two decades, compares five different farming systems: three organic (manured biodynamic, manured bioorganic, unfertilized biodynamic) and two conventional (mineral fertilized conventional, integrated conventional). The results show that organic farming increased richness, decreased evenness, reduced dispersion, and shifted the structure of the soil microbiota compared to conventionally managed soils. The impact of organic fertilizers was significant, while the plant protection regime had a subordinate effect. Systems receiving organic fertilizers harbored specific microbial guilds involved in degrading complex organic compounds, such as manure and compost. The study also found that the use of high-throughput DNA sequencing technologies can detect specific structural shifts at the individual microbial taxon level, providing new opportunities for managing soil environments by promoting beneficial and suppressing detrimental organisms.The study investigates the long-term effects of organic and conventional farming on soil microbial diversity using a high-throughput pyrosequencing approach. The DOK experiment, which has been running for over two decades, compares five different farming systems: three organic (manured biodynamic, manured bioorganic, unfertilized biodynamic) and two conventional (mineral fertilized conventional, integrated conventional). The results show that organic farming increased richness, decreased evenness, reduced dispersion, and shifted the structure of the soil microbiota compared to conventionally managed soils. The impact of organic fertilizers was significant, while the plant protection regime had a subordinate effect. Systems receiving organic fertilizers harbored specific microbial guilds involved in degrading complex organic compounds, such as manure and compost. The study also found that the use of high-throughput DNA sequencing technologies can detect specific structural shifts at the individual microbial taxon level, providing new opportunities for managing soil environments by promoting beneficial and suppressing detrimental organisms.