Bioethanol from agricultural waste is an attractive way to turn waste into added value that will solve the problem of food competition and waste management. Napier grass is a highly productive and effective lignocellulosic biomass, which is an important substrate of the second-generation biofuels. In addition, several processes are required in the production of ethanol from lignocellulosic materials; thus, co-culture fermentation can shorten the production process. This experimental research utilizes Trichoderma reesei and Saccharomyces cerevisiae co-culture fermentation in the bioethanol production of Napier grass using simultaneous saccharification and fermentation technology. To improve ethanol yield, Napier grass was pretreated with 3% (w/w) sodium hydroxide. An orthogonal experimental design was employed to optimize the Napier grass content, mixed crude co-culture loading, and incubation time for maximum bioethanol production. The results showed that pretreatment increased cellulose contents from 52.85% to 82%. The optimal fermentation condition was 15 g Napier grass, 15 g mixed crude co-culture, and 7 days incubation time, which maximizes the bioethanol yield of 16.90 g/L. Furthermore, the fermentation was upscaled 20-fold, and experiments were performed with and without supplemented sugar using laboratory-scale optimal fermentation conditions. The novelty of this research lies in the use of a mixed crude co-culture of T. reesei and S. cerevisiae to produce bioethanol from Napier grass with the maximum bioethanol concentration of 25.02 and 33.24 g/L under unadded and added sugar conditions and to reduce operational step and capital costs.