Organic C has many benefits for soil, but it is depleted by tillage and crop harvest, and especially so for biofuel crops. Accordingly, strategies such as partially retaining stover or planting a cover crop can help ameliorate the negative effect of C removal. We used a long-term field experiment to study the impacts of stover retention and planting a cover crop on soil organic matter (SOM), its extractable components, and the soil microbial community. SOM chemical composition characterization was determined by electrospray ionization (ESI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in sequential water, methanol (MeOH), and chloroform (CHCl 3) extracts. The characteristics of the soil bacterial community were measured by phospholipid fatty acid (PLFA), real-time quantitative PCR, and 16S rRNA gene sequence. The variations in total SOM content, total microbial biomass, and bacterial population were slight among treatments, but SOM chemical compounds, arbuscular mycorrhizal fungi (AMF) biomass, and bacterial structure changed significantly, and especially so in the coupled application of stover retention and cover crop. Specifically, stover retention enriched more lignin-like compounds in soil, whereas cover crop enriched more condensed hydrocarbons, and had more compounds with an aromaticity index (AI) > 0.5. The bacterial community was not altered by the cover crop, but the corn stover retention increased the relative abundances of Myxococcales (Deltaproteobacteria) and decreased that of Actinobacteria. Redundancy analysis (RDA) further revealed that the bacterial community in the stover treatments had a significant positive association with CHCl 3 -extracted chemical classes, i.e. unsaturated hydrocarbons and lipids, with the coupled application (stover and cover crop), and lignin and proteins with the corn stover only treatment. Taken together, our study shows how different C addition practices influence the molecular composition of SOM and the structure of soil microbial communities. • Soil carbon was characterized by ion cyclotron resonance mass spectrometry. • Corn stover and rye cover crop changed composition of extractable soil organic C. • Corn stover retention enhanced lignin-like compounds. • Cover crop enriched more condensed hydrocarbons. • Soil bacterial community composition corresponded to certain SOM compositions. [ABSTRACT FROM AUTHOR]