The present work discusses the influence of long-term, annually applied fertilization with manure, slurry, and NPK on the accumulation of total and available forms of phosphorus, potassium, and magnesium in soils, as well as total sulfur content. The increase in total phosphorus, potassium, and magnesium content under the influence of fertilization was significant when compared to the control group, and determined as : P-21.8%, K-36.2%, Mg-57.4%, S-28.5% in lessive soil, and P-24.7%, K-26.9%, Mg-54.2%, and S-34.7% in brown soil. An increase of available forms in the total content of macronutrients was noted. The percentage increase in relation to the control group, respectively for lessive and brown soils, was as follows: P-2.6% and 2.4%, K-1.2% and 0.6%, and Mg-0.8% (only in brown soil). A significant positive correlation between the analyzed macronutrients was confirmed in the soils, with the exception of sulfur and the available forms of phosphorus and potassium in brown soil.