A novel series of 3-hydroxyquinazoline-2,4(1H,3H)-diones derivatives has been designed and synthesized. Their biochemical characterization revealed that most of the compounds were effective inhibitors of HIV-1 RNase H activity at sub to low micromolar concentrations. Among them, II-4 was the most potent in enzymatic assays, showing an IC<sub>50</sub> value of 0.41 ± 0.13 μM, almost five times lower than the IC<sub>50</sub> obtained with β-thujaplicinol. In addition, II-4 was also effective in inhibiting HIV-1 IN strand transfer activity (IC<sub>50</sub> = 0.85 ± 0.18 μM) but less potent than raltegravir (IC<sub>50</sub> = 71 ± 14 nM). Despite its relatively low cytotoxicity, the efficiency of II-4 in cell culture was limited by its poor membrane permeability. Nevertheless, structure-activity relationships and molecular modeling studies confirmed the importance of tested 3-hydroxyquinazoline-2,4(1H,3H)-diones as useful leads for further optimization.