The use of chitosan as a flocculant has become a topic of interest over the years due to its positively charged polymer and biodegradable and non-toxic properties. However, most studies only focus on microalgae and wastewater treatment. This study provides crucial insight into the potential of using chitosan as an organic flocculant to harvest lipids and docosahexaenoic acid (DHA-rich Aurantiochytrium sp. SW1 cells by examining the correlation of flocculation parameters (chitosan concentration, molecular weight, medium pH, culture age, and cell density) toward the flocculation efficiency and zeta potential of the cells. A strong correlation between the pH and harvesting efficiency was observed as the pH increased from 3, with the optimal flocculation efficiency of >95% achieved at a chitosan concentration of 0.5 g/L at pH 6 where the zeta potential was almost zero (3.26 mV). The culture age and chitosan molecular weight have no effect on the flocculation efficiency but increasing the cell density decreases the flocculation efficiency. This is the first study to reveal the potential of chitosan to be used as a harvesting alternative for thraustochytrid cells.