AbstractBiosolids can build soil organic matter, but their ability to increase carbon and nitrogen in persistent fractions in deep soil is not well understood. We aimed to assess the mechanisms that influence soil carbon and nitrogen dynamics at three sites—Sacramento (irrigated, grazed grassland), Solano (rainfed, grazed grassland), and Merced (feed cropping system with alfalfa–corn rotation)—where soils were amended with biosolids for 20 years using density fractionations, organomineral extractions, and correlations between calcium and soil organic carbon at three depths (0–10, 30–50, and 75–100 cm). We found that amended soils had higher carbon and nitrogen content in the free‐ and occluded‐light fractions at all depths relative to the control in the Sacramento and Solano sites; however, the Merced site had a greater relative increase of carbon and nitrogen associated with the heavy fraction. Effect sizes show that biosolids increase carbon and nitrogen content in free‐ and occluded‐light fractions in the surface soil (0–10 cm), and in both light and heavy fractions in the deep soil layer (75–100 cm). Ratios of carbon to iron and aluminum show that chelation is an important mechanism of carbon stabilization in Sacramento and Solano sites throughout the soil profile. No (0–10 cm) to negative (75–100 cm) correlations were observed between calcium and soil carbon in the amended soils in the Merced site. Our results indicate that, while biosolids are typically incorporated at shallow depths, long‐term application of biosolids can increase the amount of free‐ and occluded‐light carbon fractions in deep soil.