Marine trophodynamics refer to the transfer of energy from prey to predators. In marine invertebrates and fishes, the nutrients obtained through the consumption of food and/or prey are stored as energy reserves in certain tissues and/or organs including the liver, muscle, or gonads, and that these are subsequently used as bioenergetic fuel for highly energy-demanding fundamental physiological processes. In the southern Humboldt Current System, the interaction between two highly migratory resources and top species has been observed: the swordfish (Xiphias gladius) and its prey the jumbo squid (Dosidicus gigas). Because of this trophic interaction, these species store large amounts of energy (as lipids and fatty acids) in their main organs. However, how the fatty acid profile varies in the various organs of the predator and its prey is still unknown, as is its potential use as trophic biomarkers and the ecophysiological role it plays. Our results showed a moderate similarity between the fatty acid profile of the digestive gland of D. gigas with the profiles of the liver, gonad, and muscle of X. gladius, particularly with fatty acids: palmitic (C16:0), stearic (C18:0), oleic (C18:1n9), gadoleic (C20:1), EPA (C20:5n3), and DHA (C22:6n3). Our findings on the use of fatty acids as biomarkers of the interaction between two highly migratory species in the southern Humboldt System may reveal the degree of preference swordfish have for preying on jumbo squid, particularly through the consumption of the digestive gland. In both species, a high bioenergetic fuel content characterized by a predominance of saturated, monounsaturated, and polyunsaturated fatty acids may be necessary to sustain the high energy costs involved in their migratory and reproductive processes in the Humboldt Current system.