Tuberculosis (TB) is a formidable global health concern, with millions of people developing new cases each year. TB disease is caused by the bacteria, *Mycobacterium tuberculosis* (*M. tb*). *M. tb* utilizes the Esx-1 (Esat-6 System-1) protein export system to promote virulence through the translocation of secreted proteins, or substrates. The Esx-1 protein export system is also conserved and required for virulence in the fish pathogen *Mycobacterium marinum*. As such, *M. marinum* is a widely used model system for studying Esx-1 export and therefore understanding *M. tb* pathogenesis. However, many aspects of Esx-1 biology remain uncertain. We employed genetic, biochemical, and proteomics approaches to study Esx-1 biology. We report on the links between Esx systems and the mycobacterial cell envelope, spontaneous loss-of-function mutations in Esx-1 components, novel Esx-1 substrates in *M. marinum*, and new mechanisms of Esx-1 regulation. Overall, our findings advance the field of Esx-1 research by providing insights into *M. marinum*-specific proteins as well as mechanisms of regulation worth investigating in *M. tb*. A more complete understanding of Esx biology will help inform future TB therapies and vaccines based on these complex systems.