BACKGROUND: Staphylococcus aureus bacteremia in patients with cardiac implantable electronic devices (CIED) is often associated with infective endocarditis (CIED-IE). The CIED-IE diagnosis is syndromic. Diagnostic uncertainty is common. Frequently, these patients are classified possible CIED-IE, resulting in guideline non-compliant treatment and heterogeneous outcomes. Improved outcomes require accurate diagnoses. In these patients, we evaluated whether metagenomic sequencing of microbial cell-free DNA (mcfDNA) in serial plasma specimens could improve diagnostic precision. METHODS: We studied 16 patients with staphylococcal bacteremia who were classified definite or possible CIED-IE and recommended for device removal, if there was a positive blood culture within 7 days and no concurrent deep infection. Plasma specimens obtained at consent, before extraction, and during 96 hours after extraction underwent metagenomic sequencing and quantification of staphylococcal mcfDNA. RESULTS: In 10 of 11 definite CIED-IE patients, mcfDNA persisted during antibiotic therapy for prolonged durations (median 11 days, IQR 7.5 days [7.5,15]). In these cases, mcfDNA concentration in plasma obtained early after lead extraction increased significantly and thereafter decreased rapidly. In 5 cases of possible CIED-IE, mcfDNA was undetectable after 6 days (IQR 2 days [5.5,7.5]) of antibiotic therapy and remained undetectable after CIED extraction. These mcfDNA patterns differ significantly (p=0.001), suggesting two distinct patient populations: one with definite CIED-IE and one without lead infection. CONCLUSIONS: If confirmed, these mcfDNA patterns can serve as biomarkers, together with clinical features, to improve precision in diagnosing or rejecting S. aureus CIED-IE. Strategically timed mcfDNA testing before and after CIED extraction may aid in planning therapy.