This study aim to synthesize silver nanoparticles (AgNPs) using Anthemis atropatana extract and to evaluate their chemical characteristics and antimicrobial and cytotoxic effects. The biosynthesis of AgNPs is verified using UV-visible spectrum which showing maximum absorption in 430 nm wavelength. Transmission electron microscope (TEM) and scanning electron microscope (SEM) results revealed that AgNPs has a spherical shape with an average size of 38.89 nanometres. The crystalline structure of green synthesized AgNPs in optimal conditions was confirmed by XRD analysis. The pattern of XRD peaks related to face-centred cubic (fcc) (111), (200), (220), (311) and (222) observed. Also, FTIR results verified the AgNPs synthesis using plant extract. In biological tests, the MTT results indicate the dose dependence of cytotoxic effects of AgNPs on colon cancer cell lines (HT29). The AgNPs had maximum cytotoxicity on HT29 cancer cell line at 100 μg/ml concentration, which were statistically significant comparing control cells (p < .001). Moreover, real time PCR and flow cytometry results confirmed the apoptotic effects of AgNPs. According to the results, it seems that the green synthesis of AgNPs is an eco-friendly and cost effective approach. This research provides insight into the development of new anticancer and antibacterial agents.