The composition of soil organic matter is considered to have a key influence on C sequestration and global climate change and can be associated with changes in vegetation cover in the terrestrial ecosystem. Our study aimed to evaluate the soil chemical structures and various organic components from available or reactive to more stable forms in forest soils affected by acidification and after conversion from fairly close to natural beech (Fagus sylvatica) stands to a spruce (Picea abies) monoculture. Our results revealed that the beech stands had higher contents of dissolved organic carbon and low molecular mass organic acid compared to the spruce stands. The aliphatic CH groups within the soluble alkaline-extractable organic substance (AEOS) gradually disappeared with deeper soil horizons under both forest species, while the presence of aliphatic CH groups in the low-solubility AEOS was more pronounced in the A horizon under spruce and relatively increased with depth under beech stands. The carboxylic groups were more prevalent in deeper soil horizons, while polysaccharide chains and nitrogen functional groups decreased with depth under both forest stands but were more prevalent under beech than under spruce stands. These findings suggest that the stability of organic matter through the forest soil profiles increased due to the transformation of various organic compounds from litter to more stable organic matter with higher amounts of lignin components to greater amounts of carboxylic groups and aromatic groups in deeper soil horizons. Furthermore, a higher number of mobile components of soil organic matter and carboxylic acids, together with lower pH and cation exchange capacity under spruce, resulted in the leaching of nutrients, releasing risk elements into the soil solution and accelerating the podzolization process.