Multiple combinations of antiretroviral drugs have remarkably improved the treatment of HIV-1 infection. However, life-long treatments and drug resistance are still an open issue that requires continuous efforts for the identification of novel antiviral drugs. Background: The reverse transcriptase-associated ribonuclease H (RNase H) hydrolyzes the HIV genome to allow synthesizing viral DNA. Currently, no RNase H inhibitors (RHIs) have reached the clinical phase. Therefore, RNase H can be defined as an attractive target for drug design. Objective: Despite the wealth of information available for RNase H domain, the development of RHIs with high specificity and low cellular toxicity has been disappointing. However, it is now becoming increasingly evident that reverse transcriptase is a highly versatile enzyme, undergoing major structural alterations to complete its catalysis, and that exists a close spatial and temporal interplay between reverse transcriptase polymerase and RNase H domains. This review sums up the present challenges in targeting RNase H encompassing the challenges in selectively inhibiting RNase H vs polymerase and/or HIV-1 integrase and the weak antiviral activity of active site inhibitors, probably for a substrate barrier that impedes small molecules to reach the targeted site. Moreover, the focus is given on the most recent progress in the field of medicinal chemistry that has led to the identification of several small molecules as RHIs in the last few years. Conclusion: RHIs could be a new class of drugs with a novel mechanism of action highly precious for the treatment of resistant HIV strains.