AbstractCurrently, nanotechnology is gaining massive attention compared to conventional methods as the biosynthesis of plant-based nanoparticles is considered safe, effective, and ecofriendly. Therefore, keeping in view the importance of nanotechnology, the present study was designed to synthesize, characterize, and evaluate the biological effectiveness of saffron stigma-based gold nanoparticles (SS-AuNPs) for theirin vitroandin vivobiological properties. These gold nanoparticles were characterized by UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). The highest antibacterial effect was observed by the saffron extract againstEscherichia coli(22 mm). SS-AuNPs significantly inhibited the activity of enzyme urease (54.98%) and CA-II (64.29%). However, the nonsignificant inhibitory effect was observed in the case of α-chymotrypsin. Maximum analgesic (84.98%) and antiinflammatory (88.98%) effects were observed for SS-AuNPs (10 mg/kg). Similarly, SS-AuNPs demonstrated a significant (P< 0.01) sedative effect at all tested doses.