Origanum species are known to be powerful antioxidants due to the secondary metabolites they contain. It is therefore hypothesized that nanoparticles synthesized by origanum species may have applications in medicine, cosmetics, and food production. This study aimed to investigate the antioxidant and antimicrobial activities of silver nanoparticles obtained at different times (60, 120, and 180 min) and temperatures (30, 60 and 90°C) using O. onites extract. The initial synthesis of Ag Nanoparticles (AgNps) was characterized by UV- spectrophotometry and the surface plasmon resonance band was observed at 430-450 nm under all conditions. The highest absorbance was observed at a wavelength of 425 nm for 180 min at 90°C. The FTIR spectrum showed a peak in the 3.000-3.500 cm-1 range compared to the control, confirming the reduction of silver nitrate. When the particle distribution of AgNPs prepared at three different reaction times was examined by SEM, it was observed that AgNPs with very similar particle sizes were formed. The average particle size was determined to be 171 nm, 169 nm, and 152 nm, respectively, for reaction times of 60, 120, and 180 minutes at 90°C, based on the results of the EDX analysis. The highest DPPH scavenging activity was observed in nanoparticles obtained at the third hour of the 60°C and 90°C reactions. AgNPs obtained at all 3 temperatures showed antimicrobial activity on S. aureus, E. coli, and C. albicans, however, the highest activity was found in AgNPs synthesized at 60°C and 90°C at the 3rd hour. Nanoparticles synthesized from natural materials should not display toxic properties. The AgNPs produced in this study did not exhibit toxicity towards healthy human fibroblasts. The results demonstrated that the AgNPs synthesized within the scope of this study have potential applications in biomedical fields. It was determined that the AgNPs with the highest activity were synthesized at temperatures of 60°C and 90°C at the 3rd hour.