Background: The degradative activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), termed ribonuclease H (RNase H), which hydrolyzes the RNA component of the heteroduplex RNA:DNA replication intermediate, is an excellent target for drug discovery. Anthraquinones (AQs) and their derivatives, which are common secondary metabolites occurring in bacteria, fungi, lichens and a large number of families in higher plants, have been reported to have several biological activities including that of inhibiting HIV-1 RT activities in biochemical assays. Methods: We have assayed new AQ derivatives on HIV-1 RNase H activities in biochemical assays. Results: Six series of new AQ derivatives with various substituents at positions 1, 2, 3 and 4 of the AQ ring were tested, and new analogs able to inhibit HIV-1 RT-associated RNase H activity in the low micromolar range were found. Conclusions: Our results demonstrate that AQ derivatives are promising anti-RNase H inhibitors.