Removal of biomass for bioenergy production may decrease soil organic carbon. While perennials or cover‐cropped grains often have greater root production than annual grain crops, they variably impact soil carbon and underlying mechanisms remain unclear. We used high‐frequency measurements of soil respiration and natural abundance carbon stable isotopes to differentiate respiration sources, pool sizes, and decomposition rate constants during a 10 month incubation of soils collected to 1 m depth from a 10 year old field experiment in Iowa, United States. Conversion of corn–soybean rotations to reconstructed prairies or addition of a rye cover crop to continuous corn significantly altered respiration sources and dynamics of fast‐ and slow‐cycling carbon (turnover times of weeks to months–years, respectively), but had little effect on bulk soil carbon and several extractable pools (except in fertilized prairie). Both unfertilized and fertilized prairies increased slow‐cycling carbon pools relative to annual crops, but only in 0–25 cm soil. Compared with fertilized prairie, the unfertilized prairie significantly increased decomposition rates of fast‐ and slow‐cycling carbon pools in 0–25 cm soil, likely explaining the lack of significant bulk soil carbon accrual despite twofold greater root production. Carbon derived from C4 plants decomposed faster than C3‐derived carbon across all depths and cropping systems and contributions of C3‐carbon to respiration increased with depth. Respiration of cover crop‐derived carbon was greatest in 0–25 cm soil but comprised >25% of respiration below 25 cm, implying a disproportionate impact of the cover crop on deep soil metabolism. However, the cover crop also increased the decomposition rates of fast‐ and slow‐cycling carbon pools and decreased their pool sizes across all depths relative to corn without a cover crop. Despite their notable environmental benefits, neither unfertilized perennials nor cover crops necessarily promote rapid soil carbon sequestration relative to conventional annual bioenergy systems because of concomitant increases in decomposition.