Although mucosal-associated invariant T (MAIT) cells recognize bacterial riboflavin precursor metabolites, MAIT cell stimulation by broad bacterial families and mammalian cells suggests the existence of novel ligands from different biological sources. Here, we established a comparative platform of functional metabolomics and used Mycobacterium tuberculosis as a model to characterize novel metabolites for MAIT cell activation. We extracted and fractionated small metabolites of M. tuberculosis using high-performance liquid chromatography, showing a different MAIT cell stimulation pattern of M. tuberculosis metabolite fractions in comparison with Escherichia coli fractions. Mass profiling predicted multiple nucleoside analogs enriched in a biologically active fraction of M. tuberculosis. However, the synthetic forms of some predicted M. tuberculosis nucleosides were unavailable. Further structural-based autodocking of analogous nucleosides that are conserved in mammals supported potential binding with the MR1 protein. Indeed, functional assays of these conserved nucleosides demonstrated guanosine as a stimulator and deoxyformyluridine as an inhibitor of MAIT cell activation. Identification of bioactive nucleoside metabolites broadly conserved in bacterial and mammalian systems will facilitate understanding the roles of MAIT cells against infections, inflammations, and cancers.