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The green biosynthesis of nanoparticles by plant extracts is an attractive and promising technique for medicinal applications. In the current study, we chose one of the daisy plants, <i>Aaronsohnia factorovskyi</i> (which grows in the Najd region, Saudi Arabia), to investigate its anti-microbial efficacy, in combination with silver nanoparticles. The biosynthesized nanoparticles were evaluated for antibacterial activity against <i>Staphylococcus aureus, Bacillus</i><i>subtilis</i> (Gram-positive), <i>Pseudomonas aeruginosa,</i> and <i>Escherichia coli,</i> (Gram-negative) using the disc diffusion method, while the antifungal activity was assessed against <i>Fusarium oxysporum, Fusarium solani, Helminthosporium</i><i>rostratum, and Alternaria</i><i>alternata.</i> The potential phytoconstituents of the plant extracts were identified by Fourier-transform infrared spectroscopy (FT-IR) techniques, the Field emission scanning electron microscopy (FE-SEM), Chromatography/Mass Spectrometry (GC-MS) techniques, and Zeta potential analysis. The current study revealed the ability of the tested plant extract to convert silver ions to silver nanoparticles with an average diameter of 104–140 nm. Biogenic <i>Aaronsohnia factorovskyi</i>-silver nanoparticles (AF-AgNPs) showed significant antibacterial activity against <i>Staphylococcus aureus</i> with inhibition zone diameter to 19.00 ± 2.94 mm, and antifungal activity against <i>Fusarium solani,</i> which reduced the growth of fungal yarn to 1.5 mm. The innovation of the present study is that the green synthesis of NPs, which is simple, cost-effective, provides stable nano-materials, and can be an alternative for the large-scale synthesis of silver nanoparticles.