Melanoma is the most dangerous type of skin cancer because of its high invasion and metastasis ability. Bromodomain-containing protein 4 (BRD4), an acetylation-recognizing reader, mediates the proliferation, metastasis, and invasion of melanoma, and is thus a potential therapeutic target. Mounting evidence suggests that inhibition of single bromodomain of BRD4 would improve specificity and reduce cytotoxicity to non-tumor tissues or cells. In this study, a hierarchical virtual screening campaign was performed against BRD4 BD2 from a chemical database including over 90,000 natural/natural-like compounds. Using various biochemical assays, the 7-methoxycoumarin derivative N13 was identified as a potent inhibitor of BRD4 BD2. Compared with the well-known BRD4 inhibitor JQ1, N13 exhibited higher potency against BRD4 BD2 and much higher specificity for BRD4 BD2 over BRD4 BD1. Additionally, N13 inhibited the proliferation of two kinds of BRD4-overexpressing melanoma cell lines. Mechanistically, N13 impaired the protein–protein interaction (PPI) between BRD4 BD2 and its acetylated ligand proteins (Twist1 K73/K76Ac and FOXO3a K242/245Ac), leading to reducing levels of Wnt5A and CDK6 expression, inducing cell senescence of melanoma cancer cells, and ultimately weakening the adhesion, metastasis, and invasion ability of melanoma cancer cells. To our knowledge, N13 is the first 7-methoxybicoumarin-based BRD4 BD2 inhibitor described to date and may function as a new scaffold for developing more specific and potent therapeutic agents against BRD4 BD2. In addition, our research highlights the druggability of BRD4 BD2 as a target for BRD4-overexpressing melanoma and provides a potential mechanism for the anti-melanoma activity of BRD4 BD2 inhibitor.