Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.