Aptamers are short single-stranded DNA or RNA sequences that fold into complex three-dimensional structures capable of binding to specific targets in the same manner as antibodies. Due to their small size, aptamers can penetrate tumourous masses at a much more effective rate than therapeutic antibodies. Having generated aptamers against two well recognised cancer cell surface markers, EpCAM and CD133, and shown that these aptamers are efficiently internalised via receptor-mediated endocytosis, we sought to functionalise these aptamers into targeted therapeutic agents. We intercalated a common chemotherapeutic, doxorubicin, into the stem of the aptamer and tested its effectiveness to prevent the formation of colorectal tumour spheres, an indication of metastatic ability. Both the sphere size and number were significantly smaller/reduced following treatment with our novel therapeutic. When HT29 colorectal cancer cells were treated ex vivo and injected subcutaneously into mice, no mouse formed tumours, in contrast to those treated with free drug. Additionally, following an in vivo three-day treatment schedule of HT29 xenograft tumour, we showed a significant reduction in tumour size. When the cells from these tumours were serially transplanted into additional mice, 80% of mice failed to form a tumour, even when 1,000,000 cells were transplanted, indicating that we have ‘knocked out’ the tumourigenic potential of these cancer cells. These results show the potential of our aptamer-doxorubicin conjugate to potentially eradicate the tumourgenic/cancer initiating cells within a tumourous mass, and prevent relapse. Importantly, the results in this study were achieved using a 4-fold lower concentration of doxorubicin to that used in the clinical setting, suggesting that this targeted delivery system could improve the therapeutic index of doxorubicin, and reduce the detrimental side effects experienced by cancer patients. Therefore, the combination of aptamers with conventional drugs will likely represent one of the new arsenals against cancer.