Amphiphilic kanamycins derived from the classic antibiotic kanamycin have attracted interest due to their novel bioactivities beyond inhibition of bacteria. In this study, the recently described 4″,6″-diaryl amphiphilic kanamycins reported as inhibitors of connexin were examined for their antifungal activities. Nearly all 4″,6″-diaryl amphiphilic kanamycins tested had antifungal activities comparable to those of 4″,6″-dialkyl amphiphilic kanamycins, reported previously against several fungal strains. The minimal growth inhibitory concentrations (MICs) correlated with the degree of amphiphilicity (cLogD) of the di-substituted amphiphilic kanamycins. Using the fluorogenic dyes, SYTOXTM Green and propidium iodide, the most active compounds at the corresponding MICs or at 2×MICs caused biphasic dye fluorescence increases over time with intact cells. Further lowering the concentrations to half MICs caused first-order dye fluorescence increases. Interestingly, 4×MIC or 8×MIC levels resulted in fluorescence suppression that did not correlate with the MIC and plasma membrane permeabilization. The results show that 4″,6″-diaryl amphiphilic kanamycins are antifungal and that amphiphilicity parameter cLogD is useful for the design of the most membrane-active versions. A cautionary limitation of fluorescence suppression was revealed when using fluorogenic dyes to measure cell-permeation mechanisms with these antifungals at high concentrations. Finally, 4″,6″-diaryl amphiphilic kanamycins elevate the production of cellular reactive oxygen species as other reported amphiphilic kanamycins.