The mechanisms or host factors involved in septic thrombus or vegetation formation in Staphylococcus aureus-induced infective endocarditis (IE) are unclear. Using an experimental endocarditis rat model, here we demonstrated that S. aureus HG001-induced vegetation was composed of bacterial floes encased in aggregated platelets and surrounded by neutrophil extracellular traps (NETs). In vitro data demonstrated that platelets contribute to both biofilm and NET formation. Prophylactic administration of DNase I significantly reduced the size of vegetation induced by methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains, even though MRSA and MSSA isolates express different biofilm phenotypes and NET-induction abilities in the presence of platelets. Moreover, delivery of both DNase I and daptomycin prophylactically and therapeutically produced synergistic effects by reducing vegetation size and bacterial numbers on damaged valve tissues in MRSA-induced IE. Together, these data suggest that NETs contribute to vegetation formation in S. aureus endocarditis and DNase I has the potential to control S. aureus-induced IE in the clinic.