Mycobacterium tuberculosis is the etiological agent of human tuberculosis (TB), a disease responsible for more than one million deaths in 2013. A major mechanism by which this bacterial pathogen mediates infection is through the export of virulence factors from the ESAT-6 System-1 (ESX-1). To better understand mycobacterial pathogenicity, we studied the basic genetic and molecular requirements of ESX-1 secretion and virulence in pathogenic mycobacteria. Using Mycobacterium marinum as a model species, we screened a near-saturating transposon-insertion library for mutant strains exhibiting morphology defects. We report here the identification and characterization of two mutant M. marinum strains that are deficient for ESX-1 function and attenuated in infection models. From these analyses we expanded the genetic requirements for ESX-1 secretion in M. marinum to include MMAR_4414 and the MMAR_1663-MMAR_1668 locus. We also determined that ESX-1-dependent virulence is mediated through the proteins substrates associated with the bacterial surface, contrary to the assumption that true exo-proteins facilitate virulence. Finally, we determined that the protein product of MMAR_4414 directly interacts with EccA, an established component of the ESX-1 system. In summary, these studies identified two major genetic loci required for ESX-1 function and provide novel insights into the working model of ESX-1 secretion in pathogenic mycobacteria. The continued study of these, and other yet still undiscovered ESX-1 components, may contribute to the future control and prevention of M. tuberculosis infection.