Accumulating evidence suggests that breast cancer originates from cancer stem cells (CSCs), which comprise a small percentage of the overall tumor but are highly tumorigenic and pluripotent with unlimited proliferation potential. Furthermore, CSCs are highly resistant to conventional treatment, which may explain certain difficulties in treating cancer with current therapy options. In this study, the third generation oncolytic herpes simplex virus (oHSV) vector G47 Delta effectively killed different subtypes of breast cancer cells, with more than 98% of the tumor cells killed by Day 5. Moreover, G47 Delta targeted equally non-cancer stem cells (NCSCs) and CSCs which showed resistance to paclitaxel. We demonstrated that G47 Delta effectively replicated and spread among CSCs. G47 Delta also impaired the self-renewal ability of CSCs, as the viable cells were unable to form secondary tumor spheres. We also showed that G47 Delta was able to induce the regression of tumor xenografts in BALB/c nude mice and demonstrated the ability of G47 Delta to synergize with paclitaxel by killing both NCSCs and CSCs, suggesting that oHSV may be an effective treatment modality for patients with breast cancer.