Electrochemotherapy has demonstrated significant therapeutic potential for breast cancer treatment in recent years. This study investigates the potential therapeutic effects of high-voltage nanosecond pulsed electric fields (nsPEFs) on triple-negative breast cancer (TNBC). In this study, human breast cancer cells (MDA-MB-231) were selected as the main experimental subjects. Tumor cells in vitro were treated with nsPEFs (5 ns, 50 kV/cm). Microscopy and transmission electron microscopy were used to observe the changes in tumor morphology and internal cell structure after nsPEFs stimulation. Fluorescent probes were used to observe the changes in mitochondrial membrane potential, reactive oxygen species (ROS) and calcium ion (Ca2+) concentration. And Cell Counting Kit-8 (CCK-8) kit was used to detect the cell viability after nsPEFs and drug treatment. The pulse spectra obtained by fast Fourier transform demonstrated that the pulsed electric fields with pulse widths less than 10 ns had abundant high-frequency components and thus has strong penetration. nsPEFs acted on the cell membrane while damaging the mitochondria, which was mainly manifested by the swelling of mitochondria, the disappearance of the ridges and the decrease of the mitochondrial membrane potential. The intracellular concentrations of ROS and Ca2+ were elevated with the sustained action of the pulsed electric fields. Moreover, nsPEFs increasing the tumor cells sensitivity to cisplatin while destroying the tumor cells. The findings suggest that nsPEFs could offer a promising new approach to treating TNBC, providing insights into their potential application in clinical settings and contributing to the broader field of cancer therapy innovation.