Mass spectrometry data files of whole cell lipid extracts from strains of Mycobacterium tuberculosis with engineered replacement of the gene Rv3378c, the complemented Rv3378c deletion, and the non-mutated parental strain H37Rv. Biological quadruplicate samples from each strain were profiled to assess terpene nucleoside biosynthesis. These data were analyzed in the manuscript, “Mycobacteria that cause tuberculosis have retained ancestrally acquired genes for the biosynthesis of chemically diverse terpene nucleosides” (https://doi.org/10.1371/journal.pbio.3002813) and were included in analyzed form (DOI 10.5281/zenodo.8096634). Instrument and chromatography metadata and an annotated sample list are included with the centroided data files in mzXML format.