Phagocytic cells ingest and destroy bacteria efficiently and in doing so ensure the defense of the human body against infections. Phagocytic amoebae represent a powerful model system to study the intracellular mechanisms ensuring destruction of ingested bacteria in phagosomes. Here, we discovered the presence of a bacteriolytic activity against in cellular extracts from The bacteriolytic activity was detected only at a very acidic pH mimicking the conditions found in phagosomes. It was also strongly decreased in extracts of KO cells that were previously described to kill inefficiently internalized bacteria, suggesting that the activity observed is involved in killing of bacteria in phagosomes. We purified a fraction enriched in bacteriolytic activity where only 16 proteins were detected and focused on four proteins selectively enriched in this fraction. Three of them belong to a poorly characterized family of proteins exhibiting a DUF3430 domain of unknown function and were named BadA (Bacteriolytic A), BadB, and BadC. We overexpressed the BadA protein in cells, and the bacteriolytic activity increased concomitantly in cell extracts. Conversely, depletion of BadA from cell extracts decreased significantly their bacteriolytic activity. Finally, in cells overexpressing BadA, bacterial killing was faster than in parental cells. Together these results identify BadA as a protein required for cellular bactericidal activity. They also define a new strategy to identify and characterize bactericidal proteins in cells.