A series of 1,2,3-triazolyl 3-hydroxy-quinazoline-2,4(1H,3H)-diones was constructed utilizing Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) method. The biological significance of the novel synthesized quinazolines was highlighted by evaluating them in vitro for antiviral activity, wherein several compounds exhibited excellent activity specifically against vaccinia and adenovirus. Especially, 24b11 displayed the most potent inhibitory activity against vaccinia with an EC50 value of 1.7μM, which was 15 fold than that of the reference drug Cidofovir (EC50=25μM). 24b13 was the most potent compound against adenovirus-2 with an EC50 value of 6.2μM, which proved lower than all the reference drugs. Preliminary structure-activity relationships were also discussed. To the best of our knowledge, no data are present in the literature on antiviral activity of 3-hydroxy-quinazoline-2,4(1H,3H)-diones against DNA-viruses. Thus, these findings warrant further investigations (library expansion and compound refinement) on this novel class of antiviral agents.