Bi-magnolignan (BM), a novel compound isolated from Magnolia Officinalis leaves, exhibits significant anti-tumor activity in vitro. However, the underlying mechanism remains elusive. This study examines the anti-tumor properties of BM and its mechanism of action, specifically through its interaction with BRD4, a key regulator in oncogene transcription and genome stability. Molecular docking and biolayer interferometry assay (BLI) collectively demonstrate that BM exhibits strong binding affinity to the bromodomain (BD) region of BRD4. Cellular thermal shift assay (CETSA) results confirm that BM binding increases the thermostability of BRD4, providing further evidence of the interaction between BM and BRD4. RNA-seq analysis and western blotting reveal that BM abolishes the G2/M DNA damage checkpoint and disrupts homologous recombination (HR) repair mechanisms. To explore the downstream effects of BRD4, we performed gene set enrichment analysis (GSEA) using RNA-seq data. The results indicate that BM significantly inhibits BRD4 function, leading to the downregulation of various BRD4 target genes at the transcriptional level, including MYC. Importantly, overexpression of BRD4 rescues cells from BM-induced apoptosis, DNA damage, disrupted G2/M checkpoint, and HR deficiency (HRD), highlighting the specificity of BM for BRD4. Furthermore, in vivo experiments demonstrate that BM effectively suppresses tumor growth. Collectively, these findings underscore the potential of BM as a novel and potent BRD4 inhibitor, suggesting promising prospects for the development of targeted anti-tumor therapies that specifically inhibit BRD4.