The antioxidant effects of oil-in-water nanoemulsion based on edible citrus peel essential oils on the fatty acid composition of rainbow trout fillets stored at 4 ± 2°C are investigated. Fish fillets are treated with nanoemulsion and stored for 16 days. Lipid samples are converted into fatty acid methyl esters which are then detected by gas chromatagrophy (GC). The results show that palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), vaccenic acid (C18:1ɷ-7), oleic acid (C18:1ɷ9), eicosenoic acid (C20:1ɷ9), linoleic acid (C18:2ɷ6), linolenic acid (C18:3ɷ3), eicosapentaenoic acid (EPA) (C20:5ɷ3), and docosahexaenoic acid (DHA) (C22:6ɷ3) are the most important fatty acids in fish meat. While polyene index and hypocholesterolemic:hypercholesterolaemic fatty acid ratios decrease in trout fillets during cold storage, thrombogenicity index and atherogenicity index generally increase (especially in control and Tween 80 groups). The concentrations of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) are higher in the treatment groups and the saturated fatty acids (SFAs) are lower in all groups compared to those of the control group. Application of nanoemulsion based on citrus essential oils prevents oxidation of PUFA especially EPA and DHA, thus has potential as a preservative for fish oil. Practical Applications: In recent years, nanotechnological applications have been increasingly applied to the protection of food. Similarly, natural essential oils are used to increase the shelf life of foods. This study demonstrates the combined effect of a new method of nanoemulsions and essential oils on the safety of foods.