Coffee, one of the most popular food commodities and beverage ingredients worldwide, is considered as a potential source for food industry and second-generation biofuel due to its various by-products, including mucilage, husk, skin (pericarp), parchment, silver-skin, and pulp, which can be produced during the manufacturing process. A number of research studies have mainly investigated the valuable properties of brewed coffee (namely, beverage), functionalities, and its beneficial effects on cognitive and physical performances; however, other residual by-products of coffee, such as its mucilage, have rarely been studied. In this manuscript, the production of bioethanol from mucilage was performed both in shake flasks and 5 L bio-reactors. The use of coffee mucilage provided adequate fermentable sugars, primarily glucose with additional nutrient components, and it was directly fermented into ethanol using a Saccharomyces cerevisiae strain. The initial tests at the lab scale were evaluated using a two-level factorial experimental design, and the resulting optimal conditions were applied to further tests at the 5 L bio-reactor for scale up. The highest yields of flasks and 5 L bio-reactors were 0.46 g ethanol/g sugars, and 0.47 g ethanol/g sugars after 12 h, respectively, which were equal to 90% and 94% of the theoretically achievable conversion yield of ethanol.