Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enyzmes by the outer membrane. Here we show that the type VI secretion system (T6SS) of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyze peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa utilizes specific periplasmically-localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active T6SS, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.