Methicillin-resistant Staphylococcus aureus (MRSA) cause skin-infections and life-threatening necrotizing pneumonia due to the secretion of pore-forming toxins, such as Panton-Valentine leukocidin (PVL). PVL primarily kills host macrophages and is thought to promote S. aureus immune evasion. Whether PVL merely punches holes into the plasma membrane of macrophages or activates host cell death pathways remains controversial. To identify host factors that promote PVL toxicity, we have generated recombinant PVL and performed whole genome CRISPR screen in human macrophage cell lines. This identified complement receptor, C5aR1, as a target of PVL. To validate the role of C5aR1, we have generated CRISPR mutants in human induced pluripotent stem cell-derived macrophages. In addition, we uncovered that PVL specifically interacts with distinct host lipids. Surprisingly, these lipids are enriched on different intracellular membranes in host cells, rather than the plasma membrane. To gain a spatial-temporal view on how PVL kills, we performed live-cell imaging and single-cell analysis to assess the membranes targeted by PVL, organellar health and activation of host factors. This suggests that PVL kill macrophages by targeting different intracellular membranes and activating several host cell death factors.