Sensititre YeastOne (SYO) is an affordable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method for antifungal susceptibility testing. In this study, MICs of yeast isolates from 1,214 bloodstream infection episodes, generated by SYO during hospital laboratory activity (January 2005 to December 2013), were reanalyzed using current CLSI clinical breakpoints/epidemiological cutoff values to assign susceptibility (or wild type [WT]) to systemic antifungal agents. Excluding Candida albicans (57.4% of all isolates [n = 1,250]), the most predominant species were Candida parapsilosis complex (20.9%), Candida tropicalis (8.2%), Candida glabrata (6.4%), Candida guilliermondii (1.6%), and Candida krusei (1.3%). Among non-Candida species (1.9%), 7 were Cryptococcus neoformans and 17 other species, mainly Rhodotorula species. Over 97% of Candida isolates were WT to amphotericin B and flucytosine. Susceptibility/WT rates to fluconazole, itraconazole, and voriconazole were 98.7% in C. albicans, 92.3% in C. parapsilosis complex, 96.1% in C. tropicalis, 92.5% in C. glabrata, 100% in C. guilliermondii, and 100% (excluding fluconazole) in C. krusei. The fluconazole-resistant isolates were 6 C. parapsilosis complex, 3 C. glabrata, 2 C. albicans, 2 C. tropicalis, and 1 Candida lusitaniae. Of non-Candida isolates, 2 C. neoformans were non-WT to fluconazole, whereas Rhodotorula isolates had elevated azole MICs. Overall, 99.7%-99.8% of Candida isolates were susceptible/WT to echinocandins, but 3 isolates were nonsusceptible (either intermediate or resistant) to caspofungin (C. albicans, C. guilliermondii, and C. krusei), anidulafungin (C. albicans and C. guilliermondii), and micafungin (C. albicans). Yet, including the intrinsically resistant non-Candida isolates, the echinocandin nonsusceptibility reached 1.8%. In summary, the SYO method proved to be able to detect yeast species showing antifungal resistance or reduced susceptibility.