Quantifying changes in soil microbial biomass and mineralizable C and N is important in understanding the dynamics of the active soil C and N pools. Our objectives were to quantify long-term and seasonal changes in soil organic C (SOC), soil microbial biomass C (SMBC) and N (SMBN), and mineralizable C and N in continuous sorghum [Sorghum bicolor (L.) Moench] and sorghum-wheat (Triticum aestivum L.)/soybean [Glycine max (L.) Merr.] sequences under conventional tillage (CT) and no tillage (NT) with and without N fertilization. A Weswood silty clay loam (fine, mixed, thermic Fluventic Ustochrept) in south-central Texas was sampled after planting in April, during flowering in June, and following sorghum harvest in August. More crop residue C input was retained as SOC and SMBC under NT than under CT. Soil organic C, SMBC, SMBN, and mineralizable C and N were greatest at a depth of 0 to 50 mm under NT. Mineralizable C and SMBC averaged 18% greater in rotation than in monoculture, probably due to greater C input via crop roots and residues in rotation and a shorter fallow. Mineralizable N with N fertilization was 36% greater in continuous sorghum but not different in rotated sorghum. Mineralizable C and SMBC increased an average of 5%, but mineralizable N decreased 41% from planting to flowering, probably due to rhizodeposition. From planting to post-harvest, mineralizable C and SMBC increased 9% but mineralizable N decreased 15% due to crop residue addition. Soil N availability was reduced by plant additions in the short term but enhanced in the long term.