Tillage and crop rotation/intensity can influence soil biological properties and relevant soil processes including C sequestration. This study determined the effects of long-term (25 years) no till (NT) and conventional tillage (CT) management and cropping sequence [continuous wheat (CW; Triticum aestivum L.) and a rotation of sorghum ( Sorghum bicolor L. Moench), wheat and soybean (RW; Glycine max L. Merr)] on soil microbial community structure and labile and recalcitrant microbial bio-products in central Texas. Fatty acid methyl ester (FAME) profiles, microbial biomass (MB-C, -N and -P), hot water extractable soil carbohydrates (HWE-SC) and easily extracted- (EE-) and total-glomalin-related soil proteins (T-GRSP) were analyzed. Principal component analysis of the FAME data indicated that crop management modified and selected microbial populations. In general, NT-RW resulted in the greatest richness and biodiversity of the total microbial community, soil organic C, MB-P, HWE-SC, EE- and T-GRSP. No tillage increased labile and more recalcitrant bio-products, soil organic C and total N compared to CT. The soil microbial biomass C:N:P ratio, an indicator of ecosystem nutrient limitation, suggested that the CT-RW treatment may have a soil P limitation, which was not observed in the other treatments. The treatments preferentially selected for different microbial communities, which generated microbial products that significantly influenced soil C and N retention. Our results suggested that NT in conjunction with crop rotation (RW) can be recommended for increased soil C sequestration.