The production of value-added products from microorganisms, such as single-cell protein (SCP), through the valorization of agricultural byproducts enhances circular economy while offering alternative solutions for waste treatment. In this study, SCP was obtained through the biotechnological treatment and valorization of cheese whey, the main byproduct of the dairy industry, for the development of novel edible films. To the best of the authors’ knowledge, this is the first report examining SCP as a biopolymer for edible film production. Specifically, Kluyveromyces marxianus, which has gained QPS and GRAS status, strain EXF-5288 cultivated in deproteinized cheese whey (DCW) lactose (10.0 g/L) in a 3 L fed-batch bioreactor, resulting in a SCPmax of 2.63 g/L with a protein content of up to 49.1% w/w. The addition of increased glycerol concentrations (30, 40, and 50% w/w of dry cells) as plasticizers was examined to develop SCP-based edible films. Regarding physicochemical characterization, increased glycerol concentration significantly increased moisture content (MC%) and solubility (S%), but there was not a significant difference in other parameters. Regarding wettability, SCP-based films could be described as oleophilic surfaces since the degree of oil contact angle (OCA) ranged between 46.7° ± 1.3 and 54.0° ± 0.5. The proposed holistic approach could contribute to the development of sustainable packaging materials through waste treatment.