Seasonal variations of soil organic matter (SOM) were studied in the unfertilized plot (U) and in the NPK+farmyard manure plot (NPK+FYM) of the 88-year-old ‘Static Experiment’ at Bad Lauchstadt (Germany). Decreases in the C concentrations by 0.24% (U) and 0.43% (NPK+FYM) between June and August were observed which were significant at the p < 0.01 level. The largest differences in N concentrations were 0.035% (U: August vs. September) and 0.029% (NPK+FYM: April vs. May). The C/N ratios were lowest in July and August (∼12). The seasonal variations of SOM contents were reflected in significant differences in the C concentrations of organo-mineral particle-size fractions. The proportions of soil C, associated with clay increased from 56% and 38% in April to 69% and 48% in October in the untreated and NPK+FYM-treated plot, respectively. Pyrolysis-field ionization mass spectra of whole soil samples taken in June and August showed larger differences in the molecular composition of SOM in the untreated plot than in the NPK+FYM plot. On the basis of thermograms for six important compound classes of SOM, seasonal variations in (a) their amounts and (b) their incorporation in thermally different stable humic and/or organo-mineral bonds were visualized. Within four weeks of a net mineralization of SOM, portions of phenols, lignin monomers, lignin dimers, alkylaromatics, lipids, N-containing compounds and carbohydrates reached a higher thermal stability, which can be explained by advanced crosslinking. These results represent the first application of this novel methodology to the subtle and difficult problem of seasonal SOM variations.