Many studies report organic carbon stabilization by clay minerals, but the effects of land use and soil type on the properties of humic acids (HAs) are missing. The aim of the paper is to determine the effects of land use and soil types on the characteristics of HAs, which have a considerable influence on organic matter quality. It was hypothesised that the effect of the land use on HAs properties depends on the particular size distribution. The research was performed in three ecosystems: agricultural, forest, and meadow, located in Slovakia. From each of them, the samples of 4 soil types were taken: Chernozem, Luvisol, Planosol, and Cambisol. The soil samples were assayed for the content of total organic carbon (TOC) and the particle size distribution. HAs were extracted with the Schnitzer method and analysed for the elemental composition, spectrometric parameters in the UV-VIS range, and hydrophilic and hydrophobic properties, and the infrared spectra were produced. The research results have shown that the properties of HAs can be modified by the land use and the scope and that the direction of changes depends on the soil type. The HAs of Chernozem and Luvisol in the agri-ecosystem were identified with a higher “degree of maturity”, as reflected by atomic ratios (H/C, O/C, O/H), absorbance coefficients, and the FT-IR spectra, as compared with the HAs of the meadow and forest ecosystem. However, as for the HAs of Cambisol, a higher “degree of maturity” was demonstrated for the meadow ecosystem, as compared with the HAs of the agri- and forest ecosystem. The present research has clearly identified that the content of clay is the factor determining the HAs properties. Soils with a higher content of the clay fraction contain HAs with a higher “degree of maturity”.