Background: Metagenomic next-generation sequencing of microbial cell-free DNA (mcfDNA) allows for noninvasive pathogen detection from plasma. However, there is little data describing the optimal role for this assay in real-world clinical decision making. Methods: We performed a single-center retrospective cohort study of adult patients for whom a mcfDNA (Karius©) test was sent between May 2019 and February 2021. Clinical impact was arbitrated after review and discussion of each case. Results: A total of 80 patients were included. The most common reason for sending the assay was unknown microbiologic diagnosis (78%), followed by avoiding invasive procedures (14%). The test had a positive impact in 34 (43%), a negative impact in 2 (3%), and uncertain or no impact in 44 (55%). A positive impact was observed in solid organ transplant recipients (SOTR, 71.4%, p = 0.003), sepsis (71.4%, p = 0.003), and those receiving antimicrobial agents for less than 7 days prior to mcfDNA testing (i.e., 61.8%, p = 0.004). Positive impact was driven primarily by de-escalation of antimicrobial therapy. Conclusion: Clinical impact of mcfDNA testing was highest in SOTR, patients with sepsis and patients who had been on antimicrobial therapy for less than 7 days. Positive impact was driven by de-escalation of antimicrobial therapy which may highlight a potential role for mcfDNA in the realm of stewardship. Key Points This is a retrospective study evaluating the clinical impact of mcfDNA testing at a single center. mcfDNA positively impacted clinical care in 43% of cases. Patients admitted with sepsis, patients receiving antibiotics for less than 7 days, and solid organ transplant recipients derived the most benefit from mcfDNA testing.