T cell recognition of Mycobacterium tuberculosis (Mtb)-specific peptides presented on major histocompatibility complex class I and II (MHC-I/II) contributes to immunity to tuberculosis (TB), but the principles that govern the presentation of Mtb antigens on MHCs are incompletely understood. We hypothesized that addressing this knowledge gap would accelerate TB vaccine development for use in diverse human populations. We utilized mass spectrometry (MS) analysis to identify the repertoire of peptides presented on MHCs by Mtb-infected primary human phagocytes. We revealed that substrates of Mtb’s type VII secretion systems (T7SS) are overrepresented among Mtb-derived peptides presented on MHC-I. Quantitative, targeted MS showed that Mtb’s ESX-1 secretion system is required for the presentation of Mtb antigens on MHC-I. This system is notably absent in attenuated mycobacterial strains currently used for immunization against TB. We next established a biochemical workflow for the identification of antigens presented on MHC-II. These studies revealed Mtb protein antigens that could be presented on MHC-II by human phagocytes expressing a range of MHC-II alleles. We leveraged these discoveries to develop a workflow to evaluate new vaccine candidates for their capacity to generate peptide-MHC complexes in human antigen-presenting cells that are identical to those generated during Mtb infection, a potentially critical pre-clinical screening step. Our study identifies Mtb antigens presented on MHCs that could serve as targets for TB vaccines and reveals potential explanations for the limited efficacy of existing TB vaccines in human populations.