Biochemical composition of both intracellular (biomass) and extracellular soil organic matter was determined after extraction with 0.5 M K 2SO 4. Extractable carbon, hexoses, pentoses, total reducing sugars, ninhydrin-reactive nitrogen (NRN), proteins and DNA content were colorimetrically determined. The objective of the pilot study was to examine the information potential included in newly measured biochemical characteristics, their environmental variance and the relationships with main soil properties. Correlation analysis and PCA showed independence between biochemical parameters and physico-chemical properties of the soil. Thus, the parameters characterising biochemical composition of the soil biomass and extracellular matter seem to bring new information about the soils beyond the physico-chemical parameters. They also seem to reveal a more detailed view on microbial biomass or extracellular organic matter pool than C bio or C ext alone, respectively. The variance, which occurred in biochemical characteristics, also displayed a high discrimination potential between the defined soil categories. Three types of indices were newly proposed: index I (“substrate quantity index”)—the biomass-specific amount of the extracellular organic compounds, index II (“immobilisation ratio”)—the portion of the organic compound immobilised in microbial biomass, and index III (“substrate quality index”)—the extracellular organic compound content related to extracellular organic carbon. The indices displayed a higher potential than both soil biotic and abiotic parameters to discriminate soil characters and soil types.