In the Castelporziano Reserved Area (Tyrrhenian coast near Rome, Italy), the organic horizons of 14 forest sites were chemically characterized to assess the European reference base of humus classification in Mediterranean ecosystems and evaluate the impacts of typical ecological disturbances on humification processes. Soils were sampled in three diagnostic horizons OL (litter), OF (fulvic) and OH (humic), and the biological type of humus were investigated on a morpho-functional basis and assigned to the taxonomic levels Mull, Amphi and Moder. For each diagnostic horizon, the total organic carbon (TOC%), the C/N ratio, the extractable carbon (TEC%), the humic and fulvic acids carbon (CHA%, CFA%), the degree (DH%) and the index of humification were determined. All data were statistically analyzed (ANOVA, post hoc HSD Tukey’s). In the OF horizon of some OL/OF sites, the CHA values were found very high, indicating the presence of a not-detected OH horizon. Conversely, part of the sites sampled with a complete humus profile, did not show differences between OF and OH, which should thus be grouped into a single diagnostic horizon. The humification parameters showed a fairly good correlation with the humus forms, limited to the first taxonomic level and the OF horizon. Overall, several discrepancies were found between humus classification and the chemical fingerprint of the humic profiles. In this regard, the use of screening analytical methods, i.e., the chemical characterization of OM, may provide a powerful support in modeling the morpho-functional references of humus forms on the peculiar features of Mediterranean forest environments.