FT-IR, FT-Raman, and1H-NMR spectroscopies were applied to investigate molecular changes in soil organic matter (SOM) treated with different biomasses. The experiment consisted of several plots of soil treated over a 22-year period (from 1972 to 1994) with different amendments: cattle manure (CM), cow slurries (CS), and crop residues (CR). Samples taken from these plots were analyzed and compared with the unamended soil (C), which was used as the control sample. The samples were analyzed for both total organic carbon (TOC) and total nitrogen (N) content, both at the beginning of the study and after 22 years of treatments. The plots amended with CS and CR and the unamended plots showed a decrease in TOC and N after 22 years when compared with the control at the beginning of the experiment. However, the amended soil with CM showed a linear increase in TOC and N in relation to the control.The above mentioned spectroscopic procedures were employed to characterize both the unfractionated SOM and the low molecular weight (LMW) fraction extracted in an acidic medium. This spectroscopic analysis revealed that the composition of the soils amended with CM varied significantly in relation to the other amendments in the sense that the more aliphatic and aromatic moieties are resistant to the degradation. This is corroborated by the spectroscopic analysis of the LMW fraction, which is richer in small aromatic and aliphatic carboxylic acids in the soil amended with CM. The results shown in this work reveal that the SOM formed during the amendment with CM might be more resistant to the process of decomposition.