In this study, the influence of humus substances on soil structure of six soils (Haplic Chernozem, Mollic Fluvisol, Eutric Fluvisol, Rendzic Leptosol, Eutric Cambisol, and Haplic Luvisol) of different ecosystems (forest, meadow, urban, and agro-ecosystem) in Slovakia was compared. The influence of the fractional composition of humus substances on the proportion of the fractions of water-resistant aggregates in different ecosystems was assessed. The fractions of free humic acids and those bound with monovalent cations and mobile R2O3 (r = 0.387; P < 0.05 and r = 0.415; P < 0.01), humic acids bound to the mineral components of the soil and stabile R2O3 (r = 0.477; P < 0.01 and r = 0.396; P < 0.01), and fulvic acids bound to the mineral components of the soil and stabile R2O3 (r = 0.504; P < 0.01 and r = 0.333; P < 0.05), had the most positive influence on the formation of larger (1-2 and 2-3 mm) waterresistant macro-aggregates. The amount of the extracted humus substances was the highest in agro-ecosystem > urban ecosystem > meadow ecosystem > forest ecosystem.