- Increasing human population and extreme consumption of fossil fuels create the potential to generate alternative energy sources. Bioethanol is an alternative and renewable energy resource for fossil fuels, which can be generated from low-cost raw materials. The objective of the study is to optimize pretreatment and culture conditions to enhance bioethanol production from Azolla filiculoides substrate using Saccharomyces cerevisiae. The A. filiculoides was collected from fresh-water ponds, cleaned, dried, milled to fine powder and then autoclaved for 15 min at 121 ℃. When A. filiculoides substrate was pre-treated with different acids and alkaline separately and alcohol production was monitored, significantly higher alcohol (0.1%) was produced with 1 M H2SO4, thus it was selected as the best pre-treatment agent.