© 2017 Jenkins, Swenson, Lau, Rocha, Aaring, Hazen, Chakraborty and Northen. Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the < 1 kDa fraction). This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.