We investigated the composition of soil-extracted solubilized organic and inorganic matter (SESOM) prepared from three different soils. Growth of various bacterial strains in these soil extracts was evaluated to find appropriate conditions for ecophysiological approaches. Analysis of SESOM by (1)H-NMR and gas chromatography/mass spectrometry revealed a complex mixture of organic compounds. An oak forest SESOM supported the growth of several gram-positive and gram-negative soil-derived heterotrophic bacteria, whereas beech forest and grassland soil extracts did not. A metabolomic approach was performed by determining the extracellular metabolite profile of Bacillus licheniformis in SESOM. The results demonstrated that determination of the organic composition of SESOM during batch culturing is feasible. This makes SESOM amenable to studying the ecophysiology of a range of soil bacteria growing on soil-dissolved organic matter under more defined laboratory conditions. SESOM may also increase success in isolating previously uncultured or novel soil bacteria. Cell populations and the corresponding extracellular medium can be obtained readily and specific components extracted, paving the way for proteomic, transcriptomic, and metabolomic analyses. The synthetic carbon mixture based on SESOM, which mimics soil abilities, shows a positive impact on higher cell yields and longer cultivation time for biotechnological relevant bacteria.