Roots and associated microbes generate acid-forming CO2 and organic acids and accelerate mineral weathering deep within Earth’s critical zone (CZ). At the Calhoun CZ Observatory in the USA’s Southern Piedmont, we tested the hypothesis that deforestation-induced deep root losses reduce root- and microbially-mediated weathering agents well below maximum root density (to 5 m), and impart land-use legacies even after ~70 y of forest regeneration. In forested plots, root density declined with depth to 200 cm; in cultivated plots, roots approached zero at depths >70 cm. Below 70 cm, root densities in old-growth forests averaged 2.1 times those in regenerating forests. Modeled root distributions suggest declines in density with depth were steepest in agricultural plots, and least severe in old-growth forests. Root densities influenced biogeochemical environments in multiple ways. Microbial community composition varied with land use from surface horizons to 500 cm; relative abundance of root-associated bacteria was greater in old-growth soils than in regenerating forests, particularly at 100–150 cm. At 500 cm in old-growth forests, salt-extractable organic C (EOC), an organic acid proxy, was 8.8 and 12.5 times that in regenerating forest and agricultural soils, respectively. The proportion of soil organic carbon comprised of EOC was greater in old-growth forests (20.0 ± 2.6%) compared to regenerating forests (2.1 ± 1.1) and agricultural soils (1.9 ± 0.9%). Between 20 and 500 cm, [EOC] increased more with root density in old-growth relative to regenerating forests. At 300 cm, in situ growing season [CO2] was significantly greater in old-growth forests relative to regenerating forests and cultivated plots; at 300 and 500 cm, cultivated soil [CO2] was significantly lower than in forests. Microbially-respired δ13C-CO2 suggests that microbes may rely partially on crop residue even after ~70 y of forest regeneration. We assert that forest conversion to frequently disturbed ecosystems limits deep roots and reduces biotic generation of downward-propagating weathering agents.