Dengue virus (DENV) infectious disease is a major public health problem worldwide; however, licensed vaccines or specific antiviral drugs against this infection are not available. To identify novel anti-DENV compounds, we screened 1280 pharmacologically active compounds using focus reduction assay. Bromocriptine (BRC) was found to have potent anti-DENV activity and low cytotoxicity (half maximal effective concentration [EC50], 0.8–1.6 μM; and half maximal cytotoxicity concentration [CC50], 53.6 μM). Time-of-drug-addition and time-of-drug-elimination assays suggested that BRC inhibits translation and/or replication steps in the DENV life cycle. A subgenomic replicon system was used to verify that BRC restricts RNA replication step. Furthermore, a single amino acid substitution (N374H) was detected in the NS3 protein that conferred resistance to BRC. In summary, BRC was found to be a novel DENV inhibitor and a potential candidate for the treatment of DENV infectious disease.