Oxidative stress is a potential factor in the promotion of endothelial dysfunction. In this research, flavonoids (quercetin, luteolin) combined with carotenoids (lycopene, lutein), especially quercetin-lycopene combination (molar ratio 5:1), prevented the oxidative stress in HUVEC cells by reducing the reactive oxygen species (ROS) and suppressing the expression of NADPH oxidase 4 (Nox4), a major source of ROS production. RNA-seq analysis indicated quercetin-lycopene combination downregulated inflammatory genes induced by HO, such as IL-17 and NF-κB. The expression of NF-κB p65 was activated by HO but inhibited by the quercetin-lycopene combination. Moreover, the quercetin and lycopene combination promoted the thermostability of Sirtuin 1 (SIRT1) and activated SIRT1 deacetyl activity. SIRT1 inhibitor EX-527 attenuated the inhibitory effects of quercetin, lycopene, and their combination on the expression of p65, Nox4 enzyme, and ROS. Quercetin-lycopene combination could interact with SIRT1 to inhibit Nox4 and prevent endothelial oxidative stress, potentially contributing to the prevention of cardiovascular disease.