Dietary intervention with ω-3 marine fatty acids may potentially modulate inflammation and oxidative stress markers related with CVD, metabolic syndrome and cancer. The aim of this study was to evaluate whether different proportions of ω-3 EPA and DHA intake provoke a modulation of the production of lipid mediators and then, an influence on different indexes of inflammation and oxidative stress in a controlled dietary animal experiment using Wistar rats. For such scope, a lipidomic SPE-LC-ESI-MS/MS approach previously developed was applied to determine lipid mediators profile in plasma samples. The effect of ω-3 fatty acids associated to different ratios EPA:DHA was compared with the effect exerted by ω-3 ALA supplementation from linseed oil and ω-6 LA from soybean oil. CRP showed a tendency to greater inflammatory status in all ω-3-fed animals. Interestingly, ratios 1:1 and 2:1 EPA:DHA evidenced a noteworthy healthy effect generating a less oxidative environment and modulating LOX and COX activities toward a decrease in the production of proinflammatory ARA eicosanoids and oxidative stress biomarkers from EPA and DHA. In addition, the ability of 1:1 and 2:1 fish oil diets to reduce lipid mediator levels was in concurrence with the protective effect exerted by decreasing inflammatory markers as ω-6/ω-3 ratio in plasma and membranes. It was also highlighted the effect of a higher DHA amount in the diet reducing the healthy benefits described in terms of inflammation and oxidative stress. Results support the antiinflammatory and antioxidative role of fish oils and, particularly, the effect of adequate proportions EPA:DHA.