In the current work, humic acids (HAs) isolated from natural compost and unamended and amended soils in a medium-time field experiment were characterized to evaluate the effects of the amendment at rates of 124, 239 and 478 t/ha on their chemical, compositional and structural features. The impact of the application of compost on their properties was observed over 3 years. Humic acids were characterized using spectral methods and elemental analysis. Humic acid isolated from compost was predominantly aliphatic, with a larger content of nitrogen and low degree of aromaticity and humification. The typical maximum (280/345 nm) of HA obtained from compost lies within the T (tryptophan-like) region, which can be ascribed to proteinaceous organic materials. On the other hand, the HAs obtained from amended soil were mainly aromatic in character, with a larger distribution of oxygen-containing functional groups, molecular weight and greater aromaticity. Fluorophores of HAs obtained from amended soil lie within the C (humic-like) region with typical maxima centred in the range 430–450/500–540 nm, occurring usually in HAs isolated from soil, peat and lignite. According to ultra-violet/visible and Fourier-transform infrared (FTIR) spectroscopy, the larger oxygen contents of these HAs are associated with the substitution of aromatic rings by oxygen-containing functional groups such as carboxylic, hydroxyls and ethers. On the basis of FTIR spectra, it was shown that HAs obtained from amended soil 2 and 3 years after compost application were enriched by peptid, aromatic and polysaccharide compounds absorbing at 1540, 1515 and 1040/cm, respectively.