Scutellaria multicaulis is a medicinal herb which has been extensively prescribed in Iranian traditional medicine for treatment of infections, constipation, wounds, and also as medication for anxiety. To evaluate biological activities of biosynthesized silver nanoparticles (Sm-AgNPs), Sm-AgNPswere synthesized using S. multicaulis stem extract as capping and reducing agent. Characterization studies using UV-vis, XRD, DLS, Zeta potential, FESEM, EDX, TEM, FTIR, RAMAN and TGA assays indicated that Sm-AgNPs had optimal shape, size (∼60 nm) and stability (-46.4 mV) to be used as a drug. Findings also demonstrated that Sm-AgNPs display higher Total Phenolics and Total Flavonoids Content and possesses higher antioxidant activity. HPLC-MS analysis of constituents in the S. multicaulis stem extract and Sm-AgNPs-free supernatant in negative ion mode showed that flavonoids, mainly jaceidin, skullcapflavon II, wogonin, oroxylin A and dihydroxy, trimethoxyflavone from extract have contributed to the synthesis of Sm-AgNPs. Additionally, Sm-AgNPs demonstrated effective anticarcinogenic properties on MDA-MB231 cells proliferation with IC value of 81.2 μg/mL at 48 h by inducing cell apoptosis. Overall, results confirmed the potential role of S. multicaulis stem extract as a potential raw material for synthesis of biologically active Sm-AgNPs, for development of new antitumor agents which could be utilized to combat breast cancer.