Background: Nanotechnology is receiving greater attention these days as a result of its applications in numerous industrial, medical, and environmental fields. Objective: To synthesize silver nanoparticles with a green alga, Cladophora glomerata , and determine their inhibitory activity against tumor cell (MCF-7) and transgenic mouse cell (L20B) lines. Materials and Methods: Methanol extract was prepared from Cladophora glomerata and used as a safe factory for the synthesis of silver nanoparticles (AgNPs). UV-visible spectrophotometer, X-ray diffraction, scanning electron microscopy, and EDX analyses were used to characterize the biosynthesized AgNPs. The anti-tumor activity of the phycosynthesized AgNPs was tested against the MCF-7 and L20B cell lines. Furthermore, the bioactive compounds in the algal extract were determined by gas chromatography-mass spectroscopy (GC-MS). Results: The phycosynthesis produced clusters of spherical and polydispersed cuboidal pure AgNPs with an average size of 32 nm. The phycosynthesized AgNPs possess anti-cancer and anti-tumor activities on the MCF-7 and L20B cell lines, with significant anti-proliferation percentages of 52.8 and 65.8%, respectively, after 48 hours of treatment with 100 μg/ml AgNPs. Both treated cell lines showed a significant change in cellular shape and tissue detachment. The GC-MS analysis revealed the presence of a high proportion of octadecanoic acid (47.59%) and hexadecanoic acid (14.97%). Conclusion: Cladophora glomerata contains chemicals that improve the stabilization and reduction properties of the nanoparticles. It can be used as a safe, local, and natural source for the synthesis of AgNPs and can also be used as a benign factory for many other metal nanoparticles. The phycosynthesized AgNPs have anti-cancer and anti-tumor activities on the test cell lines and provide an insight into the potential for using them as a trend in cancer nanotherapy.