Compounds that generate reactive oxygen species (ROS) have been of particular interest to those who are developing new cancer therapeutics, as current literature has established the tendency of high ROS concentrations to trigger apoptosis of cancer cells. Thus, this study aims to compare two structurally similar anti-cancer and ROS-generating 1,4-naphthoquinones, menadione and plumbagin, to bridge the gap in literature by exploring the non-enzymatic generation of ROS by these compounds to explain their anti-cancer natures and how substitution on the naphthoquinone moiety influences ROS production. Menadione and plumbagin did not produce a significant amount of non-enzymatic cleavage in the presence of reducing agents NADH, glutathione, or sodium ascorbate. However, menadione generated DNA damage through photochemical generation of hydroxyl radicals at both 350 and 419 nm, and singlet oxygen at 350 nm. Plumbagin photosensitized DNA damage only in the presence of NADH and produced ROS at lower levels at both wavelengths.