Background: Recent studies suggest that numerous naturally occurring agents have the potential to kill cancer cells via mitochondrial dysfunction. Solanum nigrum is a herb widely used in alternative medical systems. This study aimed to investigate the cytotoxic effect of Solanum nigrum water extract (SNWE) against Michigan Cancer Foundation-7 (MCF-7) and MD Anderson-Metastatic Breast Cancer-231 (MDA-MB-231) cells. Methods: We used an MTT reduction assay for cytotoxicity analysis. To explore the mode of action, the cellular adenosine triphosphate (ATP) levels and mitochondrial membrane potential were analyzed using a colorimetric ATP assay and Rhodamine-123 fluorescent staining, respectively, during SNWE treatment for 72 h. Results: The cytotoxic effect was significant in both cell lines, with IC{}_{50} values of 4.26 µg/mL and 5.30 µg/mL in MCF-7 and MDA-MB-231 cells, respectively. The 24, 48, and 72 h treatments of 100 µg/mL SNWE showed 0.85 \pm 0.07, 0.38 \pm 0.1, and 0.20 \pm 0.1 nM ATP in MCF-7 cells and 0.94 \pm 0.07, 0.84 \pm 0.2 and 0.46 \pm 0.2 nM in MDA-MB-231 cells, respectively. The SNWE treatment altered the mitochondrial membrane potential (\mathrm{\Delta }\mathrm{\Psi }m) in a concentration-dependent manner in both the breast cancer cell lines, to 29.6 \pm 4.1% in MCF-7 and 28.7 \pm 4.17% in MDA-MB-231 cells, when compared with healthy mitochondria (100% \mathrm{\Delta }\mathrm{\Psi }m). Conclusions: The cytotoxic effects of Solanum nigrum against breast cancer cells are associated with energy metabolism. Additional studies are warranted to test the anticancer effect of Solanum nigrum using an animal model of breast cancer.