Soy isoflavones have been reported to inhibit breast cancer (BC) progression; however, the underlying mechanism remains unclear. Previous studies have primarily focused on individual components of soy isoflavones. This study integrates network pharmacology, molecular docking, molecular dynamics simulations, experimental validation, and Mendelian randomization (MR) to systematically evaluate the effects and mechanisms of soy isoflavones on BC. A total of 190 potential anti-BC targets for six active soy isoflavone components were identified from multiple public databases. Enrichment analysis revealed that these targets play critical roles in regulating cellular oxidative stress and modulating drug sensitivity in BC. Ten hub targets were identified through protein-protein interaction (PPI) network analysis and topology screening: TP53, SRC, ESR1, EGFR, PIK3CA, HSP90AA1, PRKACA, HRAS, AKT1, and ITGB1. Molecular docking analysis demonstrated strong binding between these hub targets and the six soy isoflavone components, with PRKACA-daidzin (DA) and PRKACA-genistin (GE) exhibiting the strongest binding affinities. Molecular dynamics simulations further confirmed the stability of the binding interactions of these two complexes. Experimental validation indicated that DA and GE effectively inhibited BC progression, with their mechanism linked to the suppression of PRKACA expression. However, MR analyses did not find a causal relationship between the consumption of soy products and reduced BC risk. In conclusion, this study confirms the anti-BC potential of soy isoflavones and, for the first time, elucidates the anti-BC mechanism of soy isoflavones.