Background Rezafungin is a novel, once-weekly echinocandin. EUCAST rezafungin MIC testing has been associated with a good separation of WT and target gene mutant isolates in single-centre studies, but an unacceptable inter-laboratory MIC variation has prevented EUCAST breakpoint setting. This has been attributed to non-specific binding to surfaces across microtitre plates, pipettes, reservoirs, etc. used, as previously encountered for some antibiotics. Objectives To investigate use of a surfactant to mitigate non-specific binding of rezafungin in EUCAST E.Def 7.3 MIC testing. Methods Surfactants including Tween 20 (T20), Tween 80 (T80) and Triton X-100 (TX100) were evaluated for stand-alone or synergistic antifungal activity via checkerboard assays in combination with rezafungin. Subsequent T20 studies defined an optimized assay concentration, validated in up to four microtitre plate types for WT and fks mutant Candida strains (seven species total) and the six-strain EUCAST Candida quality control (QC) panel. Lastly, T20 inter-manufacturer variability, thermostability and best handling practices were investigated. Results T20 and T80 performed equivalently, with characteristics slightly preferable to TX100. Due to existing use in EUCAST mould susceptibility testing, T20 was pursued. An optimized concentration of 0.002% T20 normalized rezafungin MIC values across plate types for all Candida spp. evaluated, maintained differentiation of WT versus fks mutants and generated robust QC ranges. Additionally, T20 performance was consistent across manufacturers and temperatures. T20 can be reliably transferred utilizing a syringe, wide-orifice pipette tip and/or by mass. Conclusions Supplementation of RPMI (Roswell Park Memorial Institute) 1640 medium with 0.002% T20 generated a highly reproducible EUCAST yeast MIC methodology for rezafungin.