Photochemical and oxidative damages in retinal pigment epithelial (RPE) cells are key events in the pathogenesis of age-related macular degeneration. Polyunsaturated fatty acids (PUFA) and carotenoids are rich in retinal cells, and under oxidative stress leads to oxidation and release lipid mediators. We evaluated the impact of carotenoids (lutein, zeaxanthin) and docosahexaenoic acid (DHA) supplementation on RPE cells under oxidative stress. ARPE-19 cells were exposed to H2O2 after pre-treatment with lutein, zeaxanthin, DHA, lutein + zeaxanthin or lutein + zeaxanthin with DHA. The data showed H2O2 reduced cell viability and DHA content, while promoted catalase activity and certain oxidized PUFA products. Treatment with DHA enhanced omega-3 PUFA enzymatic oxidation namely, anti-inflammatory mediators such as hydroxy-DHA, resolvins and neuroprotection compared to control; the effects were not influenced by the carotenoids. Omega-6 PUFA oxidation, namely pro-inflammatory HETE (5-, 9-, 12 and 20-HETE), and isoprostanes (5- and 15-F2t-IsoP and 4-F3t-IsoP) were reduced by lutein + zeaxanthin while the addition of DHA did not further reduce these effects. We observed transcriptional regulation of 5-lipoxygenase by DHA and GPx1 and NEFEL2 by the carotenoids that potentially resulted in decreased HETEs and glutathione respectively. 4-HNE was not affected by the treatments but 4-HHE was reduced by lutein + zeaxanthin with and without DHA. To conclude, carotenoids and DHA appeared to regulate inflammatory lipid mediators while the carotenoids also showed benefits in reducing non-enzymatic oxidation of omega-6 PUFA.