This study addresses the critical demand for sustainable energy by optimizing bioethanol production, focusing on maximizing yield from the ethanologenic bacterial strain Enterobacter mori SVIDE3 through a systematic one-factor-at-a-time (OFAT) approach. Key fermentation parameters, such as temperature, pH, nitrogen source, and incubation time, were individually examined to determine the conditions that enhance bioethanol production. The optimized model supports scaling bioethanol production, underscoring the efficiency of Enterobacter mori SVIDE3 and the power of statistical design in microbial bioethanol optimization. The OFAT approach revealed Enterobacter mori SVIDE3’s strong carbohydrate fermentation potential, achieving ethanol yields of 4.25 g/L from glucose at optimum conditions of 72 hrs incubation, 35 °C, yeast extract as a nitrogen source, and pH 9.0, and 2.69 g/L from xylose at optimum conditions of 72 hrs incubation, 35 °C, yeast extract, and pH 7.0. This research contributes valuable insights into improving fermentation processes and addresses key scalability and commercial feasibility challenges associated with Enterobacter mori SVIDE3 for sustainable bioethanol production.