In the present study, ozone was introduced as an alternative approach to harvest and disrupt microalgae cells (Chlorella vulgaris) simultaneously for biodiesel production. At the optimum ozonation conditions (6.14 g·h�1 ozone concentration, 30 min ozonation time, 1 L·min�1 of ozone flowrate at medium pH of 10 and temperature of 30 °C), the sedimentation efficiency of microalgae cells increased significantly from 12.56 to 68.62. It was observed that the microalgae cells aggregated to form flocs after pretreated with ozone due to the increment of surface charge from �20 to �6.59 mV. Besides, ozone had successfully disrupted the microalgae cells and resulted in efficient lipid extraction, which was 1.9 times higher than the control sample. The extracted microalgae lipid was mainly consisted of methyl palmitate (C16:0), methyl oleate (C18:1) and methyl linolenate (C18:3), making it suitable for biodiesel production. Finally, utilization of recycled culture media after ozonation pre-treatment showed robust growth of microalgae, in which the biomass yield was maintained in the range of 0.796 to 0.879 g ·h�1 for 5 cycles of cultivation. Figure not available: see fulltext.. © 2021, Higher Education Press.