Magnesium is recognized to have pharmacological potential, and its nanoformulation is anticipated to offer significant therapeutic effects, particularly against cancer. In this study, we analyzed the anticancer effect of biogenically synthesized magnesium oxide nanoparticles (MgO NPs) against breast cancer cells (MDA-MB-231). Different biological evaluations, such as cytotoxicity, cellular morphology, induction of apoptosis, generation of ROS, cell adhesion and cellular migration were estimated using well established methodology. The biogenic MgO NPs exhibited increased cytotoxicity, induced apoptosis, enhanced formation of ROS, promoted cell adhesion and inhibited cellular migration in a dose-dependent manner, showing its therapeutic potential against MDA-MB-231 cells. The current study observed strong anticancer activity of MgO NPs against studied cancer cell lines. However, our study must be validated in an appropriate animal/xenograft model to authenticate the effectiveness of MgO NPs against breast cancer.