Triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is an aggressive breast cancer subtype with limited targeted therapeutic options. Although conventional topoisomerase II (Topo II) inhibitors, such as doxorubicin, are widely used, their efficacy is limited by multidrug resistance and dose-limiting cardiotoxicity. In this study, we evaluated MHY5945, a newly synthesized chrysin-derived flavonoid, as a Topo II-targeting anticancer agent for TNBC. MHY5945 markedly inhibited the proliferation and viability of MDA-MB-231 TNBC cells and induced G2/M cell-cycle arrest and apoptosis. Mechanistically, MHY5945 activated the DNA damage response, as indicated by increased γH2AX and Chk1/Chk2 activation, together with altered G2/M cell-cycle regulatory proteins. MHY5945 also modulated apoptosis-related proteins, consistent with the induction of apoptotic cell death following DNA damage and cell-cycle arrest. In addition, MHY5945 induced lower P-glycoprotein expression than doxorubicin, suggesting that it may be less susceptible to P-glycoprotein-mediated drug resistance. Collectively, these findings demonstrate that MHY5945 exerts potent anticancer effects in TNBC cells by inducing Topo II inhibition-associated DNA damage, G2/M cell-cycle arrest, and apoptosis. These results identify MHY5945 as a promising Topo II-targeting candidate for preclinical investigation.