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This study evaluated the fermentative performance of several yeast strains on the aqueous by-product of waste ice cream (WIC) to ethanol, comparing 3 reported lactose-utilizers, Kluyveromyces marxianus Y-329, Dekkera anomala Y-1414, and Kluyveromyces lactis 8585, with the non-lactose utilizer Streptomyces cerevisiae Y-45. Fermentation progression was monitored by weight loss, and HPLC was used to analyze the composition of the fermentates. Ethanol production reached >10% for S. cerevisiae and K. marxianus but was <5% for D. anomala and K. lactis, possibly due to catabolite repression from the hydrolysis of sucrose. Treatment of the WIC by-product with lactase increased the availability of fermentable carbohydrates, resulting in greater ethanol yields for all strains except K. marxianus Y-1414, which may be limited by its ethanol tolerance. A minimal medium supplemented with lactose, sucrose, or a combination of both sugars was used to test whether the yeast could metabolize lactose alone or in the presence of sucrose. Sucrose was utilized by all 4 strains and produced ethanol in the absence and presence of lactose. Lactose was only utilized by D. anomala Y-1414 and K. lactis 8585 in the absence of sucrose, confirming that the presence of sucrose can prevent lactose utilization by yeasts in this study. Fermentation efficiency was also determined, with S. cerevisiae Y-45 having achieved the greatest efficiency in sucrose alone, whereas K. lactis 8585 showed the greatest efficiency in lactose alone. Results from this study showed that S. cerevisiae remains the ideal candidate for the valorization of waste ice cream, as it had the highest ethanol yield in WIC by-product with lactose and increased when lactose was hydrolyzed with lactase, reaching 12.5% at 72 h.