Phagocytic cells ingest bacteria and kill them in phagosomes. A variety of molecular mechanisms allow the killing and destruction of bacteria in phagosomes, but their complete list and relative importance remain poorly defined. Here we have used Dictyostelium discoideum amoebae as model phagocytic cells. Our results reveal that PldX, a luminal phospholipase D, plays an important role in the phagosomal destruction of ingested bacteria. Analysis of bacterial destruction in wild-type and pldX KO living cells suggests that PldX participates in the permeabilization of the bacterial membrane. The bacteriolytic activity of D. discoideum extracts was also measured in vitro: extracts from pldX KO cells exhibit significantly less bacteriolytic activity than wild-type cells, confirming the role of PldX in the lysis of bacterial membranes. These results identify luminal phospholipase D as a major player in the permeabilization of bacterial membranes in phagosomes.