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The present work examined the production of single-cell protein (SCP) by a newly isolated strain of <i>Kluyveromyces marxianus</i> EXF-5288 under increased lactose concentration of deproteinized cheese whey (DCW) and different temperatures (in °C: 20.0, 25.0, 30.0 and 35.0). To the best of the authors’ knowledge, this is the first report examining the ability of <i>Kluyveromyces marxianus</i> species to produce SCP at T = 20.0 °C. Different culture temperatures led to significant differences in the strain’s growth, while maximum biomass and SCP production (14.24 ± 0.70 and 6.14 ± 0.66 g/L, respectively) were observed in the cultivation of <i>K. marxianus</i> strain EXF-5288 in shake-flask cultures at T = 20.0 °C. Increased DCW lactose concentrations (35.0–100.0 g/L) led to increased ethanol production (Eth_max = 35.5 ± 0.2 g/L), suggesting that <i>K. marxianus</i> strain EXF-5288 is “Crabtree-positive”. Batch-bioreactor trials shifted the strain’s metabolism to alcoholic fermentation, favoring ethanol production. Surprisingly,<i> K. marxianus</i> strain EXF-5288 was able to catabolize the produced ethanol under limited carbon presence in the medium. The dominant amino acids in SCP were glutamate (15.5 mg/g), aspartic acid (12.0 mg/g) and valine (9.5 mg/g), representing a balanced nutritional profile