HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations . Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants . The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor . Here, we characterize the mode of action of - (2-hydroxy-benzylidene) -3,4,5-trihydroxybenzoylhydrazone (compound), an N-acylhydrazone derivative that inhibited viral replication (EC = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus . Time-of-addition and biochemical assays showed that compound targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being> 20-fold less potent against the RT polymerase activity . Docking calculations revealed that compound binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.