Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, such as RNH, should be effective against all of the current drug-resistant variants. Here, we present 2.80 A and 2.04 A resolution crystal structures of an RNH inhibitor, beta-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. beta-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that beta-thujaplicinol is a slow-binding RNH inhibitor with noncompetitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNADNA substrate.