The current work concentrated on the green synthesis of silver nanoparticles (AgNPs) through the use of aqueous Citruslimon zest extract, optimizing the different experimental factors required for the formation and stability of AgNPs. The preparation of nanoparticles was confirmed by the observation of the color change of the mixture of silver nitrate, after the addition of the plant extract, from yellow to a reddish-brown colloidal suspension and was established by detecting the surface plasmon resonance band at 535.5 nm, utilizing UV-Visible analysis. The optimum conditions were found to be 1 mM of silver nitrate concentration, a 1:9 ratio extract of the mixture, and a 4 h incubation period. Fourier transform infrared spectroscopy spectrum indicated that the phytochemicals compounds present in Citrus limon zest extract had a fundamental effect on the production of AgNPs as a bio-reducing agent. The morphology, size, and elemental composition of AgNPs were investigated by zeta potential (ZP), dynamic light scattering (DLS), SEM, EDX, X-ray diffraction (XRD), and transmission electron microscopy (TEM) analysis, which showed crystalline spherical silver nanoparticles. In addition, the antimicrobial and antioxidant properties of this bioactive silver nanoparticle were also investigated. The AgNPs showed excellent antibacterial activity against one Gram-negative pathogens bacteria, Escherichia coli, and one Gram-positive bacteria, Staphylococcus aureus, as well as antifungal activity against Candida albicans. The obtained results indicate that the antioxidant activity of this nanoparticle is significant. This bioactive silver nanoparticle can be used in biomedical and pharmacological fields.