Mutations and reduced mitochondrial DNA (mtDNA) content are commonly observed in breast cancer, yet their functional significance is not clear. This study aimed to determine whether the mtDNA content in breast cancer plays an important role in modulating the response to anthracycline treatment in vivo and in vitro. The mtDNA content in tumor cells was analyzed using quantitative polymerase chain reaction in 60 Taiwanese breast cancer patients to correlate with their survival. In addition, human breast cancer MDA-MB-231 cells were treated with ethidium bromide to decrease mtDNA copy number. Cell survival was determined by trypan blue exclusion assay and intracellular reactive oxygen species (ROS) were determined by flow cytometry. After an anthracycline-based regimen, the disease-free survival of patients with higher mtDNA content breast cancer was significantly lower than that of patients with lower mtDNA content breast cancer (p = 0.03). Moreover, the MDA-MB-231 cells with low copies of mtDNA had higher sensitivity to doxorubicin treatment and increased ROS production when compared with higher mtDNA parental cells. Our results suggest that the level of mtDNA copy number in breast cancer may be a potential biomarker for prediction of the response to anthracycline-containing regimens in breast cancer patients.