Increasing food production while avoiding the progressive degradation of agricultural soils has become one of the major challenges at a global level. In consequence, the development of sustainable tillage methodologies or cultivation strategies is an important subject of current research. In fact, it has been observed that the implementation of reduced tillage (RT) vs. traditional tillage (TT) in the long term not only improves soil physicochemical properties but also global soil quality in terms of soil health. In particular, the increase of the soil organic carbon (SOC) content under RT conditions is one of the most important factors, but there is little information about the chemical composition and humification level of this carbon, and thus about its persistence at long-term. This is of particular importance considering the policies of carbon sequestration and climate change mitigation, such as the “4 per 1000” initiative. In this study, molecular-level characterization of the humic acid (HA) and fulvic acid (FA) fractions isolated from a soil after 19 years under RT and TT practices was carried out. This study would provide objective descriptors of the impact of these two tillage practices in the chemical composition of the resulting SOC. With this purpose, the potential of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) for assessing changes in the molecular assemblages released from the humic fractions (HA and FA) was examined. The results showed enhanced diversity and chemical richness (expressed as number of molecular formulas) in the composition of SOC under RT. Different tillage-specific compound classes were associated with both tillage practices. As a whole, the humic fraction showed a higher proportion of molecular formulas for lipid and hydroaromatic families in the case of RT compared to TT, while the same fraction under TT showed a greater richness of oxidized protein-derived formulas than RT. In the case of FAs, a similar pattern was observed for hydroaromatic and protein-derived formulas, but the proportion of molecular formulas assigned to unsaturated lipids was higher in TT than in RT. In addition, increased number of formulas for aromatic and condensed aromatic compounds was observed in FAs under TT respect to RT.