Ribonuclease H (RNase H) inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 Reverse Transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral Xenotropic Murine Leukemia Virus-Related Virus (XMRV) and Moloney Murine Leukemia Virus (MoMLV) RTs to HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than HIV-1 RT and comparable to MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn(2+) and not Mg(2+). Using hydroxyl-radical foot-printing assays we demonstrated that the distance between the polymerase and RNase H domains in MoMLV and XMRV RTs is longer than in HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with IC(50)s ranging from ∼0.8 to 0.02 μM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.