<p><i>Ganoderma resinaceum</i> Boud., Polyporaceae, is a medicinal fungus that has long been traditionally used owing to its ability to kill cancer cells. Polysaccharides derived from <i>Ganoderma</i> species, including <i>G. resinaceum</i>, have shown potential in cancer therapy due to their ability to induce apoptosis, inhibit metastasis, and modulate immune responses. This study aimed to investigate the therapeutic potential of <i>G. resinaceum</i> polysaccharides in breast cancer <i>in vitro</i>, focusing on their effects on cell cycle dynamics, apoptosis, epithelial–mesenchymal transition, and drug resistance. <i>Ganoderma resinaceum</i> polysaccharides were isolated, purified, and characterized using liquid chromatography–mass spectrometry and Fourier transform infrared spectroscopy. Their effects on cell survival, the cell cycle, apoptosis, reactive oxygen species generation, migration, invasion, and mammosphere formation were assessed in Michigan Cancer Foundation-7 (MCF-7), M D Anderson-Metastatic Breast-231 (MDA-MB-231), and doxorubicin-resistant MCF-7^Dox/R cells. Western blotting and immunophenotyping were used to analyze apoptotic and epithelial–mesenchymal transition markers. <i>G. resinaceum</i> polysaccharides exhibited selective cytotoxicity, induced cell cycle arrest, promoted apoptosis via the generation of reactive oxygen species, and suppressed migration and invasion by downregulating epithelial–mesenchymal transition markers (Slug, Zeb1, and Vimentin). Additionally, <i>G. resinaceum</i> polysaccharides impaired mammosphere formation, indicating their ability to target cancer stemness. <i>G. resinaceum</i> polysaccharides demonstrated multifaceted activity against cancer cells; thus, their ability to target both drug-sensitive and drug-resistant breast cancer cells highlights their potential as future therapeutic agents.</p> Graphical Abstract <p></p>