Innate immune responses to bacteria require cooperative interactions between host recognition molecules and phagocytes. The peptidoglycan recognition proteins (PGRPs) are a large group of proteins found in insects and mammals that bind to bacterial peptidoglycan (PGN). PGRP-S is located with other antimicrobial proteins, such as lysozyme, in the granules of human neutrophils. Whereas both PGRP-S and lysozyme recognize PGN, the exact binding specificity of human PGRP-S, its functional activity, and its potential synergy with other neutrophil-derived bactericidal proteins such as lysozyme have not been determined. Here we show that human PGRP-S binds to and inhibits the growth of Staphylococcus aureus (containing lysine-type PGN) and Escherichia coli (containing mesodiaminopimelic acid-type PGN). The binding affinity and thus antimicrobial activity of PGRP-S is determined by the third amino acid in the PGN stem peptide. Furthermore, the antimicrobial effect of PGRP-S against E coli is synergistic with lysozyme, and lysozyme and PGRP-S colocalize in neutrophil extracellular traps (NETs), suggesting that these granule-derived proteins act together to kill bacteria trapped in the NETs. Taken together, these results indicate that human PGRP-S plays a role in innate immunity in the context of neutrophils by contributing to the killing of intracellular and extracellular bacteria.