Extraction of lipid is an important step in the production of microalgae biodiesel, but its efficiency depends on the species and also the pre-treatment methods for cell disruption before undergoing extraction stage. Thus, ozonation pre-treatment were used in this study to address the knowledge gap surrounding the harvesting and pre-treatment of microalgae for efficient lipid extraction. Chlorella vulgaris was ozonized in batches using lab-scale ozone generator (HATLEA) in the range of 5 to 25 minutes where the sedimentation efficiency of microalgae was found to be optimum at 25 minutes of ozonation time and flow rate of 3 L/min. Based on these conditions, the sedimentation efficiency was 31 higher compared to non-ozonated microalgae. Besides, lipid yield was increased from 18 (non-ozonated) to 25.7 after ozonation under optimum conditions. Therefore, it can be concluded that the ozonation pre-treatment of microalgae can be applied in the overall process of microalgae biodiesel production as alternative steps to reduce the cost of production. © 2018 Author(s).