ABSTRACTRezafungin is a novel once-weekly echinocandin for intravenous injection currently in development for the treatment ofCandidainfections and the prevention ofCandida,Aspergillus, andPneumocystisinfections in allogeneic blood and marrow transplant recipients. Whilein vitrodata indicated that rezafungin exposure was unlikely to be affected by commonly prescribed medicines, interactions resulting in the altered systemic exposure of some drugs coadministered with rezafungin could not be excluded. Two phase 1 open label crossover studies, conducted in healthy subjects, examined drug interactions between rezafungin and multiple drug probe cytochrome P450 (CYP) substrates and/or transporter proteins, immunosuppressants, and cancer therapies. Statistical analysis compared the outcomes for drugs coadministered with rezafungin to those for the drugs administered alone. The geometric mean ratio was reported, and a default 90% confidence interval (CI) no-effect equivalence range of 80 to 125% was used for the maximal plasma concentration (Cmax), the area under the curve from time zero to the final sampling time point (AUC0–t), and the AUC from time zero to infinity (AUC0–∞). Most probes and concomitant drugs were within the equivalence range. For tacrolimus, ibrutinib, mycophenolic acid, and venetoclax, the AUC orCmaxwas reduced (10 to 19%), with lower bounds of the 90% CI values falling outside the no-effect range. The rosuvastatin AUC andCmaxand the repaglinide AUC0–∞were increased (12 to 16%), with the 90% CI being marginally above the upper bound. Overall, thein vitroandin vivodata demonstrated a low drug interaction potential with rezafungin via CYP substrate/transporter pathways and commonly prescribed comedications, suggesting that coadministration was unlikely to result in clinically significant effects. Treatment-emergent adverse events were typically mild, and rezafungin was generally well tolerated.IMPORTANCEAntifungal agents used to treat life-threatening infections are often associated with severe drug-drug interactions (DDIs) that may limit their usefulness. Rezafungin, a newly approved once-weekly echinocandin, has been shown to be free of DDIs based on extensive nonclinical and clinical testing described in this study.