India is among the largest banana producing countries and thus banana pseudostem is commonly available agricultural waste to be used as lignocellulosic substrate. Present study focuses on exploitation of banana pseudostem as a source for bioethanol production. In this experiment, we have used three different microorganisms, S. cerevisiae, E. coli and C. albicans, these were found to be better for ethanol production. Substrates were enzymatically saccharified by the inoculation of isolated microorganism for the production of ethanol. Optimal pH and temperature for better yield of ethanol were 5.6 to 6.5 and room temperature respectively. Autoclave pre-treatment protected the samples from contamination and increased volume of ethanol. The total sugar content and ethanol yield in the fermentation was estimated during the period (1 day interval) of fermentation and concluded that amount of total sugar content gradually decreased due to the microbial utilization( as energy source), when ethanol content increases.