Mitochondria are dynamic organelles frequently undergoing fission and fusion events to maintain their integrity, bioenergetics and spatial distribution, which is fundamental to the processes of cell survival. Disruption in mitochondrial dynamics plays a role in cancer. Therefore, proteins involved in regulating mitochondrial dynamics are potential targets for treatment. mDIVI1 is an inhibitor of the mitochondrial fission protein DRP1, which induces i) mitochondrial oxidative stress and ii) effectively reduces mitochondrial metabolism. We show here that mDIVI1 is able to inhibit 3D tumorsphere forming capacity, cell migration and stemness-related signalling in breast cancer cells, indicating that mDIVI1 can potentially be used for the therapeutic elimination of cancer stem cells (CSCs).