Cancer stem cells have been indicated in the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. In breast cancer, they may reside in the CD44+CD24-/low population. The use of oncolytic adenoviruses presents an attractive anti-tumor approach for eradication of these cells because their entry occurs through infection and they are, therefore, not susceptible to those mechanisms that commonly render stem cells resistant to many drugs. We isolated CD44+ CD24-/low cells from patient pleural effusions and confirmed stem cell-like features including oct4 and sox2 expression and Hoechst 33342 exclusion. CD44+CD24-/low cells, including the Hoechst excluding subpopulation, could be effectively killed by oncolytic adenoviruses Ad5/3-Δ24 and Ad5.pk7-Δ24. In mice, CD44+CD24-/low cells formed orthotopic breast tumors but virus infection prevented tumor formation. Ad5/3-Δ24 and Ad5.pk7-Δ24 were effective against advanced orthotopic CD44+CD24-/low-derived tumors. In summary, Ad5/3-Δ24 and Ad5.pk7-Δ24 can kill CD44+ CD24-/low, and also committed breast cancer cells, making them promising agents for treatment of breast cancer.