Abstract Background This study characterizes the clinical utility and validity of the Karius Test® (KT), a plasma microbial cell-free DNA (mcfDNA) sequencing platform, as an infection surveillance tool among hematopoietic stem cell transplant (HCT) recipients, including monitoring for cytomegalovirus (CMV) and detecting infections relative to standard microbiologic testing (SMT). Methods A prospective, observational cohort study was performed among adult HCT recipients as inpatients and outpatients. Serial KTs were performed starting with one sample within 14d pre-HCT, then weekly from 7-63 days post-transplant then monthly from 3-12 months post-HCT. Diagnostic performance of KT vs CMV PCR was evaluated with positive percent agreement (PPA) and negative percent agreement (NPA). Infectious events (<12 months post-HCT) were extracted from medical records. For infectious events without positive SMT, two clinicians adjudicated KT results to determine if any detections were a probable cause. Difference in time from KT pathogen detection and infection onset was calculated. Results Of the 70 participants, mean age was 49.9 years. For CMV surveillance, PPA was 100% and NPA was 90%. There was strong correlation between CMV DNA and KT molecules per microliter (r2: 0.84, p<0.001). Of the 32 SMT+/KT+ infectious events, KT identified 26 earlier than SMT (median: -12d) and an additional 5 diagnostically-difficult pathogens identified by KT but not SMT. Conclusion KT detected CMV with high accuracy and correlation with qPCR. Among infectious events, KT demonstrated additive clinical utility by detecting pathogens earlier than SMT and those not detected by SMT.