Background: The primary objective of this study was to test whether oncolytic herpes simplex virus type 1 (HSV1) could eradicate chemoresistant cancer stem cells (CSCs). Methods: The fluorescent aldefluor reagent-based technique was used to identify and isolate ALDH(br) cells as CSCs from the 4T1 murine breast cancer cell line. The presence of ALDH(br) 4T1 cells was also examined in 4T1 breast cancer transplanted in immune-competent syngeneic mice. Results: Compared with ALDH(lo) cells, ALDH(br) cells had a markedly higher ability to form tumor spheres in vitro and a higher tumorigenic potential in vivo. ALDH(br) cells also exhibited increased doxorubicin resistance in vitro, which correlated with a selective increase in the percentage of ALDH(br) cells after doxorubicin treatment and an increased expression of P-glycoprotein (P-gp), a known chemoresistance factor. In contrast, oncolytic HSV1 was able to kill ALDH(br) cells in vitro and even more markedly in vivo. Furthermore, in in vivo studies, systemic administration of doxorubicin followed by intratumoral injection of oncolytic HSV1 resulted in much more significant suppression of tumor growth with increased median survival period compared with each treatment given alone (p < 0.05). Though more CD8(+) T lymphocytes were induced by oncolytic HSV1, no significant specific T cell response against CSCs was detected in vivo. Conclusions: These results suggested that the use of oncolytic HSV1 following doxorubicin treatment may help eradicate residual chemoresistant CSCs in vivo.