This study is designed to evaluate soil organic matter (SOM) quality indicators: molecular indicators of dissolved organic matter (DOM) and hydrophobicity of humic acid (HA), distribution of quantity in humified and labile fractions of histosols during renaturalization. The aim is to determine the differences in the qualitative composition of humic acids at the molecular level, which are decided by the previous tillage and genesis, and to evaluate the impact of anthropogenization on the peat soil according to hydrophobicity, as well as to estimate the impact of soil genesis and removing peat layer. Soil samples were taken from the three Sapric Histosol (according to WRB2022) profiles and the 0–30 cm layer in three field replicates (Lithuania, Radviliskis mun.). Our study suggested that in the differently managed drained Sapric Histosol under renaturalization, the most significant changes occurred in the topsoil layer (0–30 cm), in which an increase in the content of SOM particles 106–2 µm in size. It is expedient to grow perennial grasses and legumes to maintain the soil organic carbon stability mobile humic acids to mobile fulvic acids ratio (MHA:MFA 0.83 to 0.86). An evaluation of the quality of HA (E4:E6) revealed their highest maturity in the unfertilized perennial grasses (3.88) and crop rotation (3.87) with grasses. The highest concentrations of hydrophilic groups (ratio of the C=O to O-H) were found in Sapric Histosol under deciduous hardwood forest (12.33). The lowest hydrophilicity (9.25 and 9.36) was of the crop rotation Sapric Histosol with removed peat layer. The most sustainable use of drained Sapric Histosol in the context of the sustainability and quality of its humus substances should be associated with the formation of perennial grass and clover grassland and the cultivation of deciduous hardwood. Therefore, the horizon forms on the top part of the profile, which protects deeper Histosolic material layers from its mineralization.