In this study, Origanum onites was used to synthesize the silver nanoparticles (AgNPs@Org). The structure of nanoparticles was identified by spectroscopic techniques. The maximum absorption was determined as 433 nm by UV-Vis spectroscopy. In Fourier-transform infrared spectroscopy spectrum, the characteristic signal was observed at 3,262 cm(-1) belonging to the OH group. The crystal structure of nanoparticles was revealed by X-ray diffraction analysis. The diffraction peaks (2 theta) can be indexed to 111, 200, 220, 311, and 222 components representing the face-centered cubic unit structure. The spherical particle size was calculated as 18.1 nm by transmission electron microscopy. Cytotoxic effects of extract and AgNPs@Org were executed by MTT assay using Capan-1, L929, and Caco-2 cell lines. AgNPs@Org exhibited the excellent cytotoxic effect on Capan-1 cell lines with the viability of 37.6% (0.5 mu g & BULL;mL(-1)). However, the effect of O. onites extract on the viability of Capan-1 cell lines was found to be 24.6% and 55.4% at 1.0 and 0.5 mu g & BULL;mL(-1), respectively. AgNPs@Org effect on Caco-2 cell lines was found as 31.7% (1.0 mu g & BULL;mL(-1)). In the L929 cell lines, the noticeable lethal influence was not detected for extract and nanoparticles. In other words, the extract and AgNPs@Org did not act a cytotoxic effect on L929 cell lines.