Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. In this paper, we report the production of mammospheres from three lines of TNBC cells and demonstrate that both parthenolide (PN) and its soluble analog dimethylaminoparthenolide (DMAPT) suppressed this production and induced cytotoxic effects in breast cancer stem-like cells, derived from dissociation of mammospheres. In particular, the drugs exerted a remarkable inhibitory effect on viability of stem-like cells. Such an effect was suppressed by N-acetylcysteine, suggesting a role of reactive oxygen species (ROS) generation in the cytotoxic effect. Instead z-VAD, a general inhibitor of caspase activity, was ineffective. Analysis of ROS generation, performed using fluorescent probes, showed that both the drugs stimulated in the first hours of treatment a very high production of hydrogen peroxide. This event was, at least in part, a consequence of activation of NADPH oxidases (NOXs), as it was reduced by apocynin and diphenylene iodinium, two inhibitors of NOXs. Moreover, both the drugs caused downregulation of Nrf2 (nuclear factor erythroid 2-related factor 2), which is a critical regulator of the intracellular antioxidant response. Prolonging the treatment with PN or DMAPT we observed between 12 and 24 h that the levels of both superoxide anion and hROS increased in concomitance with the downregulation of manganese superoxide dismutase and catalase. In addition, during this phase dissipation of mitochondrial membrane potential occurred together with necrosis of stem-like cells. Finally, our results suggested that the effect on ROS generation found in the first hours of treatment was, in part, responsible for the cytotoxic events observed in the successive phase. In conclusion, PN and DMAPT markedly inhibited viability of stem-like cells derived from three lines of TNBCs by inducing ROS generation, mitochondrial dysfunction and cell necrosis.