Protoplast fusion, which is a novel genetic engineering approach, was developed between mesophilic and thermophilic butanol producing bacteria to enhance production of biobutanol as a green energy resource. Three strains of anaerobic gram-positive clostridia were fused through a protoplast fusion technique to produce biobutanol from wheat straw as a feedstock during the process of Simultaneous Saccharification and Fermentation (SSF). These strains have the natural enzymatic ability for biobutanol production, and include Clostridium beijerinckii (ATCC BA101), Clostridium thermocellum, and Thermoanaerobacterium saccharolyticum. The objective of the present study was to increase enzymatic activity during saccharification by raising the temperature of fermentation to increase biobutanol production. Results showed that protoplast fusion of thermophilic and mesophilic clostridia have led to improving thermostability in a fermentation medium at 45°C. This represents the optimum temperature for enzymatic hydrolysis. Results also showed that the fused strain produced essential hydrolysis enzymes, which eliminated the need to add any enzymes during the hydrolysis step. Furthermore, results in the present study demonstrated that the fused culture of bacteria was able to tolerate the elevated concentration of acetone, butanol, and ethanol during production, which resulted in higher biobutanol production of 13.8 g/L. This study included a comparison to the coculture as a benchmark to account for the effects of protoplast fusion.