Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) contribute to the pathogenicity of several mycobacteria. Biosynthesis of these virulence factors requires polyketide synthases and other enzymes that represent potential targets for the development of adjuvant antivirulence drugs. We used six isogenic Mycobacterium marinum mutants, each with a different gene knockout in the PDIM/PGL biosynthetic pathway, to probe the pleiotropy of mutations leading to PDIM(-) PGL(-), PDIM(+) PGL(-) or PDIM(-) PGL(+) phenotypes. We evaluated the M. marinum mutants for changes in antibiotic susceptibility, cell envelope permeability, biofilm formation, surface properties, sliding motility and virulence in an amoeba model. The analysis also permitted us to begin exploring the hypothesis that different gene knockouts rendering the same PDIM and/or PGL deficiency phenotypes lead to M. marinum mutants with equivalent pleiotropic profiles. Overall, the results of our study revealed a complex picture of pleiotropic patterns emerging from different gene knockouts, uncovered unexpected phenotypic inequalities between mutants, and provided new insight into the phenotypic consequences of gene knockouts in the PDIM/PGL biosynthetic pathway.