Perforin-2 is a highly conserved pore-forming protein encoded by macrophage expressed gene 1 (MPEG1). A number of studies have shown that Perforin-2–deficient mice are unable to survive following a bacterial challenge that is nonlethal in WT mice. There is also recent evidence that Mpeg1+/–heterozygous mice display an intermediate killing ability compared with Mpeg1 WT and Mpeg1–/–mice. Despite these in vivo findings, to date, no perforin-2 deficiencies have been associated with human disease. Here, we report four patients with persistent nontuberculous mycobacterial infection who had heterozygous MPEG1 mutations. In vitro, neutrophils, macrophages, and B cells from these patients were unable to kill Mycobacterium avium as efficiently as normal controls. CRISPR mutagenesis validated the deleterious antibacterial activity of these mutations. These data suggest that perforin-2 haploinsufficiency may contribute to human susceptibility to infections with intracellular bacteria.