Background Humic substances (HS) are compounds with a complicated structure, present in the humus soil layer, water, lake sediments, peat, brown coal and shales. Due to their similar physicochemical properties to DNA, they may have an adverse effect on the subsequent use of the isolated material. The main aim of this research was to examine the effect of HS on DNA isolation depending on the soil type and land use, taking into account the spectroscopic full characteristics of HS fractions. Methods The research was conducted on eight types of soil sample. Soils represented the most important Soil Reference Groups for temperate climates: Fluvisols, Regosols, Cambisols, Arenosols, Histosols and Luvisols. Soil samples were also collected from areas diversified in terms of use: arable land, grassland and forest. The extraction of HS fractions was performed using the procedure recommended by the International HS Society. The fractional composition of HS was characterized by UV–Vis and fluorescence methods. Soil DNA is extracted by direct cell lysis in the using a CTAB-based method with a commonly-used commercial soil DNA isolation kit. The basis for assessing the quantity and quality of extracted DNA was the Polymerase chain reaction (PCR) reaction since the analysis of soil DNA often relies on the use of PCR to study soil microorganisms. Results Based on the results, it can be concluded that in the presence of a high concentration of HS, the isolated DNA was low quality and the additional purification procedure was necessary. Despite the differentiation of the internal structure of HS fractions, the decisive factor in the efficiency of DNA isolation from soil samples was the total carbon content in HS. Reduced DNA yields can significantly constrain PCR detection limits to levels inadequate for metagenomic analysis, especially from humus-rich soils.