Silver nanoparticles (AgNPs), synthesized by green methods with the property to kill microbes, are highly valuable in medical sciences. So, the current study was aimed at using the flower extract of Wedelia urticifolia for synthesizing AgNPs with antibacterial properties. The AgNPs were produced by adding the extract to three different AgNO concentrations (1, 10, and 100 mM) in nine possible flower extract to metal salt ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9). The formation of brown color and the presence of a peak at 431 nm in the UV-Vis spectrum of the colloidal solution indicates the synthesis of AgNPs, which were also characterized by dynamic light scattering (DLS), Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The DLS results exposed that the smallest sized AgNPs were obtained in 10 mM AgNO solution and 4E6M was the optimized extract to metal salt solution ratio. The characterization techniques revealed that the synthesized AgNPs were spherical shaped and crystalline with a diameter of less than 30 nm. Furthermore, the synthesized nanoparticles were tested against two Gram-positive (Klebsiella pneumonia and Staphylococcus aureus) and two Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacterial strains for their antibacterial efficiency. Although the studied strains showed limited growth, overall, the effect of nanoparticles was found to be insignificant. It is concluded that the current study is advantageous over other previous studies because the AgNPs were synthesized at room temperature from 10 mM AgNO concentration in only 2 hours. Additionally, the present work is simple, ecofriendly, and in-expensive.© 2020 Wiley Periodicals, Inc.