Despite significant advances in antiretroviral therapy, acquired immunodeficiency syndrome remains as one of the leading causes of death worldwide. New antiretroviral drugs combined with updated treatment strategies are needed to improve convenience, tolerability, safety, and antiviral efficacy of available therapies. In this work, a focused library of coumarin derivatives was exploited by cell phenotypic screening to discover novel inhibitors of HIV-1 replication. Five compounds (DW-3, DW-4, DW-11, DW-25 and DW-31) showed moderate activity against wild-type and drug-resistant strains of HIV-1 (IIIB and RES056). Four of those molecules were identified as inhibitors of the viral RT-associated RNase H. Structural modification of the most potent DW-3 and DW-4 led to the discovery of compound 8a. This molecule showed increased potency against wild-type HIV-1 strain (EC<sub>50</sub> = 3.94 ± 0.22 μM) and retained activity against a panel of mutant strains, showing EC<sub>50</sub> values ranging from 5.62 μM to 202 μM. In enzymatic assays, 8a was found to inhibit the viral RNase H with an IC<sub>50</sub> of 12.3 μM. Molecular docking studies revealed that 8a could adopt a binding mode similar to that previously reported for other active site HIV-1 RNase H inhibitors.