Soil organic matter (SOM) quantity and quality are important factors that significantly influence soil fertility. SOM quality indicators change throughout time. In this study, long—term field experiments (22–50 years in duration) on a Cambisol at four sites in the Czech Republic were selected. Seven crops were successively rotated in the sequence: clover, winter wheat, early potato, winter wheat, spring barley, potato, and spring barley with interseeded clover. Five treatments were investigated, including an unfertilized treatment, farmyard manure, and various combinations of farmyard manure and mineral fertilization. A total of 40 t ha−1 of farmyard manure was applied to the early potato and potato crops. Combining organic and mineral fertilizers increased soil organic matter quality and quantity over unfertilized or organic only treatment. The highest intensity of mineral fertilizers in our trials elevated the mean of carbon sequestration efficiency to 45.6% in comparison to pure manure treatment which reached only 22.9% efficiency. A strong correlation was established between the total glomalin content and soil organic matter carbon, fulvic acid, humic acid, carbon hot water extraction, potential wettability index (PWI), and aromaticity index. The PWI was also strongly correlated with these indicators. The E4/E6 ratio indicator was shown to be a much less sensitive method for reflecting the change in soil organic matter quality.