AbstractAurantiochytrium sp. strain L3W is a heterotrophic microorganism that produces docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which are essential for the growth of marine fish. In this study, pickle seasoning liquid waste was used for culturing strain L3W and the raw biomass of strain L3W was then fed to red sea bream fingerlings (Pagus major) to confirm its usability as a source of DHA and EPA. The strain L3W was cultured on the three pickle seasoning liquid waste samples, Akimurasaki, Hiroshimana, Shisohijiki, diluted with sand-filtered seawater at initial pH values of 4 and 7 under unsterile conditions. The growth of strain L3W was highest at 3.71 g/L on Hiroshimana at an initial pH of 7 with DHA and EPA production at 71.4 and 6.8 mg/g-biomass, respectively. Preparing the raw biomass of strain L3W by the 200 L-scale cultivation using the American Type Culture Collection 790By+ medium because of the DHA and EPA contents close to that produced in the Hiroshimana medium with an initial pH of 7, the raw biomass was spiked to the diet at 3%, 5%, and 10%. The final DHA and EPA contents in the whole body were increased by 4.26 and 3.03 times, respectively, by adding the strain L3W biomass at 10%. These results confirmed the feasibility of a carbon cycling scenario in which the strain L3W is cultivated using liquid food waste with the resultant biomass is utilized as a source of DHA and EPA for fish aquaculture. Graphical Abstract