A novel series of carbohydrate-based naphthoquinones was synthesized and evaluated for cytotoxicity against different cancer cell lines. The compounds derived from 5-hydroxy-1,4-naphthoquinone (juglone) showed better cytotoxicity profiles against HCT-116, A-549 and MDA-MB 435 human cancer cells than the parent compound. The results suggest that the hydroxyl group on the aromatic ring increased the pro-oxidant activity of these new naphthoquinone derivatives. Furthermore, two derivatives were found to be more active against melanoma cells (MDA-MB435) than the clinically useful anticancer agent doxorubicin, and none of the compounds caused mouse erythrocyte lysis.