The chemical properties of pine needle litter cause slow decomposition, which results in an accumulation of highly lignocellulosic material on the forest floor. Decomposition of organic matter is important for the nutrient turnover in pine forests on nutrient-poor soils. We studied the biodegradation of needles in an organic layer focusing on the various stages of lignin degradation by fungi. Samples were obtained from pine needle litter and a stratified organic layer over nutrient-poor sand under a 60-year-old Scots pine (Pinus sylvestris) forest stand. Pyrolysis mass spectrometry (PyMS) and pyrolysis gas chromatography mass spectrometry (PyGCMS) were used to characterize the chemical composition of the needles and the soil. The pyrolysis data show that diterpenoid acids are a main component in fresh needles, but rapidly decrease in the organic layer of the soil, as a result of decomposition. The chemical composition of the soil profile is dominated by guaiacyl lignin and polysaccharides from needle litter. The hexose/pentose ratio increases with depth in the soil profile. The partial preservation of hexose polymers is the result of the preferential decomposition of pentose polymers by white-rot fungi, and points to the input of microbially synthesized polysaccharides. Indications for the degradation of guaiacyl lignin are also found in the soil profile. Oxidative reactions by soil fungi result in a shortening of the side chain of the guaiacyl lignin derivatives and an increase of carbonyl and carboxyl groups. These degradational patterns of lignin in the soil profile under Scots pine are similar to those observed in lignin model compounds and wood lignin degraded by fungi under controlled laboratory conditions.