Background: Microalgae are one of the promising feedstock that consists of high carbohydrate content which can be converted into bioethanol. Pre-treatment is one of the critical steps required to release fermentable sugars to be used in the microbial fermentation process. In this study, the reducing sugar concentration of Chlorella species was investigated by pre-treating the biomass with dilute sulfuric acid and acetic acid at different concentrations 1, 3, 5, 7, and 9 (v/v). Results: 3,5-Dinitrosalicylic acid (DNS) method, FTIR, and GC-FID were employed to evaluate the reducing sugar concentration, functional groups of alcohol bonds and concentration of bioethanol, respectively. The two-way ANOVA results (p < 0.05) indicated that there was a significant difference in the concentration and type of acids towards bioethanol production. The highest bioethanol yield obtained was 0.28 g ethanol/g microalgae which was found in microalgae sample pre-treated with 5 (v/v) sulfuric acid while 0.23 g ethanol/g microalgal biomass was presented in microalgae sample pre-treated with 5 (v/v) acetic acid. Conclusion: The application of acid pre-treatment on microalgae for bioethanol production will contribute to higher effectiveness and lower energy consumption compared to other pre-treatment methods. The findings from this study are essential for the commercial production of bioethanol from microalgae. © 2019 The Author(s).