Soil conservation practices are critical for agricultural sustainability, and in this study the shotgun sequencing approach was used to investigate the effects on soil biodiversity of different soil- and crop-management practices in a 13-year field trial in southern Brazil. Treatments consisted of conventional tillage (CT) with plowing and disking, or no-tillage (NT) with direct sowing into the residues of previous crops, in a crop succession [soybean (summer)/wheat (winter)] or rotation [soybean/maize (summer)/wheat/lupine/oat (winter)]. About 1 million reads per treatment revealed very high levels of diversity. The majority of the sequences were attributed to the Bacteria (54%), and 0.3% and 0.2% fitted into Archaea and Eucarya domains, respectively; 46% showed no similarity with any known sequences. Major differences were associated with tillage and, to a lesser extent, with crop management. Statistically significant higher abundances with CT encompassed microorganisms associated with residue decomposition, carbon and nitrogen cycling, and xenobiosis. Eucarya were also more abundant with CT, possibly related to higher tolerance of environmental stresses. In contrast, NT showed higher abundances particularly of nitrogen-fixing Rhizobiales and Archaea that inhabit environments rich in organic matter.