Today, with the rapid development of nanotechnology, it is necessary to produce nanoparticles with non-toxic methods for safe use in medical applications. For this reason, silver nanoparticles obtained by green synthesis, which are simple, clean, non-toxic, cost-effective, and environmentally friendly, are preferred. In our study, silver nanoparticles were synthesized by the green synthesis method from indigo extract, which has been used as a medicinal plant for many years due to its numerous benefits such as wounds, spots, diarrhea, shortness of breath, immune system strengthening, and protective effect against cancer. Reduction of silver ions was observed resulting in the formation of highly stable silver nanoparticles in the solution as a result of the reaction of aqueous silver nitrate solution and phytochemical-rich indigo extract. The characterization of the silver nanoparticles formed as a result of the biosynthesis reaction, in which the transformation from light yellow to dark brown color was observed, was carried out using UV-vis spectroscopy and scanning electron microscopy. It was determined that silver nanoparticles showed antibacterial activity against gram-negative (Staphylococcus aureus and Escherichia coli) and gram-positive (Bacillus subtilis) bacteria by the disk diffusion method.