Soil organic matter (SOM) represents one of the largest and most important reservoirs of organic carbon in terrestrial eco-systems. The quality of SOM (e.g., content of soil organic carbon, humic substances, humic acids, fulvic acids, and HA/FA ratio) is a key factor in determining soil fertility and productivity. The loss of SOM is currently considered a detrimental form of soil degradation. The study aims to evaluate the effect of mineral fertilising (NPK), farmyard manure, cattle slurry, and digestate applications in intensively used permanent grassland. The long-term field experiments were carried out at the locality Jevíčko, region of Boskovice Furrow Malá Haná (the Czech Republic). The effect of amending soil with different organic and mineral fertilisers was studied. Qualitative parameters of SOM, soil reaction, and the content of available nutrients (N, P, K, Ca, Mg) were followed. Results showed that the control (no organic input) and the NPK variants had the lowest SOM quality. Higher SOM quality was after the farmyard manure, Slurry, and Digetsate applications. The highest acidity (indicated by the lowest values of pH/KCl) was determined in the control, NPK, Slurry, and D variants. The most favourable soil reaction and available nutrient content were after the farmyard manure application. Humic substances spectral absorption in the UV-VIS and infrared spectral ranges showed that the highest absorbance and amount of aliphatic hydrophobic components were observed after the application of farmyard manure. The multi-criteria statistical evaluation, including principal component analysis and factor analysis, confirmed that amending soil with farmyard manure and slurry resulted in an increase in humic substances content and improved SOM quality.