In the antioxidant system in cyanobacteria, non-enzymatic antioxidants, such as carotenoids, are considered good candidates for coping with oxidative stress, particularly light stress, and pharmaceutical therapeutic applications. A significant amount of carotenoid accumulation has been recently improved by genetic engineering. In this study, to achieve higher carotenoid production with higher antioxidant activity, we successfully constructed fiveSynechocystissp. PCC 6803 strains overexpressing (OX) native genes related to the carotenoids biosynthetic pathway, including OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. All of the engineered strains maintained a significant quantity of myxoxanthophyll, while increasing zeaxanthin and echinenone accumulation. In addition, higher components of zeaxanthin and echinenone were noted in all OX strains, ranging from 14 to 19% and from 17 to 22%, respectively. It is worth noting that the enhanced echinenone component responded to low light conditions, while the increased β-carotene component contributed to a high light stress response. According to the higher antioxidant activity of all OX strains, the carotenoid extracts presented lower IC50in lung cancer cell lines H460 and A549, with values less than 157 and 139 µg/mL, respectively, when compared with those of WTc, particularly OX_CrtRand OX_CrtQ. A higher proportion of zeaxanthin and β-carotene in OX_CrtRand OX_CrtQ, respectively, may considerably contribute to the ability to treat lung cancer cells with antiproliferative and cytotoxic effects.