Green synthesis methods are environmentally friendly, cost effective and nonhazardous for biomedical applications in comparison with other methods. The aim of the study was green synthesis of silver nanoparticles using medicinal animal dung extract as a reducing, capping and stabilizing agent for the first time among other synthesis methods of silver nanoparticles. Female donkey’s dung was capable to reduce silver ions to nanoparticles and stabilize them. Silver nanoparticles with average sizes of 36 nm were synthesized and characterized by UV-Vis, FT-IR, XRD and TEM. Moreover, synthesized nanoparticles were analyzed in terms of anticancer activity by MTT assay on MCF-7 cell line. UV–Visible spectrophotometer showed an absorbance peak in the range of 414-433 nm. To identify the phytochemical coating of particles, FTIR analysis was used. Transmission electron microscope (TEM) images and X-ray diffraction (XRD) confirmed the formation of small spherical silver nanoparticles. The MTT assay revealed potent anticancer effects of the aqueous extract synthesized nanoparticles on MCF-7 cells, incubated for 24 hours. Based on the current findings, it is strongly believing that the use of donkey’s dung offers large scale production of biocompatible silver nanoparticles that can be suggested to possess valuable anticancer agents against breast cancer cell lines.