Dengue virus (DENV) is an important human arthropod-borne virus with a major impact on public health. Nevertheless, a licensed vaccine or specific treatment is still lacking. We therefore screened the NIH Clinical Collection (NCC), a library of drug-like small molecules, for inhibitors of DENV replication using a cell line that contains a stably replicating DENV serotype 2 (DENV2) subgenomic replicon. The most potent DENV inhibitor in the NCC was δ opioid receptor antagonist SDM25N. This compound showed antiviral activity against wild-type DENV2 in both Hela and BHK-21 cells, but not in the C6/36 cell line derived from the mosquito Aedes albopictus. The structurally related compound naltrindole also inhibited DENV replication, albeit less potently. Using a transient subgenomic replicon, we demonstrate that SDM25N restricts genomic RNA replication rather than translation of the viral genome. We identified a single amino acid substitution (F164L) in the NS4B protein that confers resistance to SDM25N. Remarkably, an NS4B amino acid substitution (P104L), which was previously shown to confer resistance to the DENV inhibitor NITD-618, also provided resistance to SDM25N. In conclusion, we have identified a new DENV inhibitor, SDM25N, which restricts genomic RNA replication by – directly or indirectly – targeting the viral NS4B protein.