Crop richness in crop rotation systems may modulate soil microbial activity and the diversity of community structure, driving soil organic matter (SOM) content and quality. We aimed to assess the long-term effect of crop systems on SOM composition and the structure and diversity of the bacteria and archaea in no-till subtropical soil. The study was based on samples from a subtropical Ferralsol subjected to 27 years of crop succession (wheat/soybean) or crop rotation (vetch/maize/wheat/soybean). Soil samples were analyzed in relation to C and N contents, and submitted to DNA extraction for 16 S rDNA sequencing and ¹³C Nuclear Magnetic Resonance spectroscopy – solid-state (CP/MAS ¹³C NMR). The effect of the current crop species at the time of sampling on the structure and diversity of the microbial community was predominant in relation to the long-term crop history over time (crop rotation). A greater diversity and abundance of soil bacteria and archaea was observed in the area with wheat growing in winter than with soybean growing in summer, a combined effect of crop species and season. The soil under crop rotation had a greater proportion of carbohydrate-derived C in SOM, whereas a greater proportion of alkyl-C and N/O alkyl-C was observed under crop succession. The crop species richness in crop rotation allows the maintenance of soil functions related to the formation of more biochemically labile SOM and contributes positively to soil ecosystem functioning.