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Green microalga Botryococcus braunii is a eukaryotic photosynthetic microorganism considered as a rich source of biofuel compounds, such as hydrocarbons and lipids, despite its rather slow growth. To improve the biomass, hydrocarbon, and lipid productivity, an ethyl methanesulfonate-induced mutagenesis was carried out on the indigenous microalga, Botryococcus braunii AARL G036. Among those mutants, EMS-mutant E1.0H15 gave the highest biomass and hydrocarbon productivity, which was higher than 1.17, 1.45, and 1.16-fold, respectively, when compared with the wild type (WT). Subsequently, two-stage cultivation with crucial stress factors was successfully exploited to boost the biofuel productivity of EMS-mutant E1.0H15 by increasing the lipid content by 1.22-fold (up to 48.6%) and by 1.20-fold for the hydrocarbon content (up to 71.6%) compared to one-stage cultivation. More importantly, after 24-month maintenance, the high biofuel productivity in EMS-mutant E1.0H15 remains unchanged. In addition, the fatty acid composition (>96% C16–C18) and fuel properties of the mutated microalgal lipid-derived biodiesel indicated good oxidation stability (17.5 h) and high heating values (38.6 MJ kg−1), which were in accordance with those of the international standards, i.e., EN-14214 and ASTM D675140. This strategy could contribute greatly to the further development of the microalgal strain, allowing improvement and cultivation for the desired biofuel properties. [Display omitted] [ABSTRACT FROM AUTHOR]