We investigated the ability of a natural and diverse population of MGI to assimilate organic substrates using metagenomics,metatranscriptomics, and fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-nanoSIMS). We found that both MGI Shallowand Deep Clades contain and express genes for uptake or processing of organic substrates, in addition to chemoautotrophy. We observed extensive assimilation of urea- and amino acid-derived nitrogen, as well as inorganic carbon. Thaumarchaeota MGI are therefore predominantly chemoautotrophic, with organic matter used primarily to meet nitrogen demands.