Global warming has become a major concern as a result of the excessive release of greenhouse gas emissions. An important strategy for achieving carbon neutrality targets is to focus on renewable energy resources. Second generation bioethanol synthesis via sugarcane bagasse (SCB) is another promising approach for the reduction of greenhouse gas emissions. Here in, this study presents the second generation of bioethanol production from sugarcane bagasse with the pretreatment condition adjoined with basic hydrogen peroxide and pressure effect by fermentation using microorganisms Saccharomyces Cerevisiae and Bacillus Subtilis. The results revealed better production through pretreatment at different operational stages through batch fermentation. Different characterization techniques including Scanning Electron Microscopy (SEM), Fourier Transform Infra-Red (FTIR), High Performance Liquid Chromatography (HPLC), and Thermogravimetric Analysis (TGA) results confirmed the better effects of structural changes of hemicellulose, lignin, and cellulose during treatment, weight loss, thermal stability, and higher concentration of the produced bioethanol in the distillate After pretreatment, the conversion of biomass to bioethanol by using Saccharomyces Cerevisiae gives a high production yield (70%), which presents a production of 70g/L from 100g of SCB at the end of 72 h and a yield of bioethanol (0.7g/g) of SCB confirmed through gas chromatography/mass spectrometry qualitative analysis (GC/MS). The pretreatment conditions of alkaline hydrogen peroxide (H2O2) were optimized to the values 3h, 50°C, 60 psi, pH 8.6, and 150 rpm. This study sheds light on the effects of pretreatment conditions for bioethanol production from sugarcane bagasse.