Silver nanoparticles (AgNPs) were synthesized with wheat straw biomass at room temperature using light irradiation. The reaction conditions were optimized, including the light intensity, biomass concentration, NaCl addition, and reaction time. The silver nanoparticles fabricated at the optimum conditions (light intensity 60,000 lx, biomass concentration 2 mg/mL, and reaction time 90 min) were characterized by UV-Vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and zeta potential. The TEM results showed that the silver nanoparticles were mostly spherical, with an average diameter of 17.2 nm. The zeta potential of AgNPs reached -21.6 mV. The XRD spectra showed that the AgNPs were highly crystalline, with four characteristic peaks. The FTIR of nanoparticles implied that alcohols and proteins may have a vital role in the formation and stability of AgNPs. The silver nanoparticles synthesized by wheat straw biomass revealed antimicrobial activity against Escherichia coli and Bacillus subtilis strains.