The impact of conservation tillage on the composition, stability, and origin (plant- or microbial-derived) of soil organic matter (SOM) within soil profiles remains unclear. In this study, we characterized the impact of different tillage practices on the molecular composition and status of SOM. These tillage practices included conventional tillage (CT) and conservation tillage such as rotary tillage (RT) and no-tillage (NT). Our investigation spanned a two-decade period within a temperate agroecosystem. Soil samples were collected down to a 30 cm profile, and four biomarkers, namely free lipids, lignin phenols, neutral sugars, and amino sugars, in conjunction with 13C NMR techniques were used to quantify SOM characteristics. The results revealed that conservation tillage (especially NT) led to improvements in both the quantity and composition of SOM when compared to CT in the topsoil (0–5 cm), but not in deeper layers (5–30 cm). More precisely, the NT topsoil, as opposed to CT, exhibited enhancements in two categories: (1) an increase in plant-derived compounds such as long-chain lipids (≥ C20) and steroids by 51%, lignin phenols by 106%, and plant-derived neutral sugars by 61%, and (2) an augmentation in microbial-derived biomolecules, which includes microbial-derived neutral sugars by 49% and microbial necromass carbon (MNC) by 94%. Furthermore, NT, relative to CT, increased the contribution of MNC to soil organic carbon in topsoil (up to 64%, mainly fungal necromass). This highlighted the predominant role of microbial-derived biomolecules in SOM formation. Similarly, RT treatments enhanced the microbial-derived neutral sugars by 18%, plant-derived neutral sugars by 14%, and lignin phenols by 43% relative to CT in the topsoil. Collectively, our study suggests that NT practice could be considered an effective strategy for enhancing SOM accumulation and persistence by fostering the accrual of plant- and microbial-derived biomolecules in the topsoil.