Mammalian professional phagocytic cells ingest and kill invading microorganisms and prevent the development of bacterial infections. Our understanding of the sequence of events that results in bacterial killing and permeabilization in phagosomes is still largely incomplete. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte to study the fate of the bacteria Klebsiella pneumoniae inside phagosomes. Our analysis distinguishes three consecutive phases: bacteria first lose their ability to divide (killing), then their cytosolic content is altered (permeabilization), and finally their DNA is degraded (digestion). Phagosomal acidification and production of free radicals are necessary for rapid killing, membrane-permeabilizing proteins BpiC and AlyL are required for efficient permeabilization. These results illustrate how a combination of genetic and microscopical tools can be used to finely dissect the molecular events leading to bacterial killing and permeabilization in a maturing phagosome.