Using nondestructive and destructive methods such as FTIR spectroscopy and pyrolysis-field ionization mass spectrometry (Py-FIMS), we analyzed nine macromorphologically differentiated horizons of a moder profile without chemical pretreatment. Chemical changes within this humus profile are discussed, including additional external information from wet-chemical data and CPMAS 13C NMR spectroscopy. In accordance with all methods polysaccharides, uncondensed lignin building blocks, free fatty acids, and wax esters declined with advanced stages of decomposition. Moreover, results from low- and high-resolution Py-FIMS gave evidence for elemental compositions of pyrolysates that contain nitrogen, such as amines, nitriles, and heterocycles. In addition, these measurements revealed that dimeric, condensed pyrolysis products of lignin remain constant over the whole profile whereas bound n-fatty acids increase with profile depth. This allowed understanding of existing discrepancies between wet-chemical data and CPMAS 13C NMR spectroscopy concerning the amounts and the dynamics of aromatic carbon and alkyl-C and demonstrated that lignin building blocks identified after CuO oxidation are not suitable to describe the whole lignin macromolecule. Furthermore, Py-FIMS showed an increasing portion of aromatic esters derived from suberin of stems and roots with profile depth and the accumulation of furanoid structures in the Ah horizon. Therefore, the relative enrichment of individual compounds during litter decomposition and humification due to contributions from root litter, recalcitrance of individual compounds, and (bio)chemical stabilization in the form of subunits of higher molecular polymers seems to be of considerable importance for the genesis of forest humus.