Background: Silver nanoparticles (AgNPs) have been used as an antimicrobial and disinfectant agent. Nevertheless, there is limited data about antitumor potential. This study has focused on investigating cytotoxic effects of AgNPs from Scrophularia striata flower extract on MCF-7 breast cancer cells and its mechanism of action. Materials and Methods: Thus, a green method was created for the synthesis of AgNPs using an aqueous extract of S. striata flower. Synthesis of AgNPs was described by different analytical techniques including ultraviolet-visible spectrophotometer, field-emission scanning electron microscopy, X-ray diffraction, and Fourier transforms infrared spectroscopy. Cell viability was determined by the 3-[4, 5-dimethylthiazol-2-yl]-a 2,5-diphenyltetrazolium bromide assay. Reactive oxygen species (ROS) formation was measured using probe 2', 7'-dichlorofluorescein diacetate and intracellular calcium (Cai2+) was evaluated with probe flu3-AM. Cells were treated with different concentrations of AgNPs (1, 3, 6, 10, 15, 25, 50, and 100 μg/mL). Results: The results showed that AgNPs hindered cell growth in a dose-dependent manner. AgNPs appeared to have dose-dependent cytotoxicity against MCF-7 cells through activation of the ROS generation and an increase in the intracellular Cai2+ (IC50 52 ± 3.14). Conclusion: In conclusion, the results of this preliminary study demonstrated that AgNPs from S. striata flower extract may be a potential therapeutic agent for human breast cancer treatment.