Ammi visnagais a biennial or annual herbaceous plant belonging to the familyApiaceae. For the first time, silver nanoparticles were synthesized using an extract of this plant. Biofilms are a rich source of many pathogenic organisms and, thus, can be the genesis of various disease outbreaks. In addition, the treatment of cancer is still a critical drawback for mankind. The primary purpose of this research work was to comparatively analyze antibiofilms againstStaphylococcus aureus, photocatalytic activity against Eosin Y, and in vitro anticancer activity against the HeLa cell line of silver nanoparticles andAmmi visnagaplant extract. The systematic characterization of synthesized nanoparticles was carried out using UV–Visible spectroscopy (UV-Vis), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), dynamic light scattering (DLS), zeta potential, and X-ray diffraction microscopy (XRD). The initial characterization was performed with UV-Vis spectroscopy, where a peak appeared at 435 nm, which indicated the SPR band of the silver nanoparticles. AFM and SEM were performed to determine the morphology and shape of the nanoparticles, while EDX confirmed the presence of Ag in the spectra. The crystalline character of the silver nanoparticles was concluded with XRD. The synthesized nanoparticles were then subjected to biological activities. The antibacterial activity was evaluated by determining the inhibition of the initial biofilm formation withStaphylococcus aureususing a crystal violet assay. The response of the AgNPs against cellular growth and biofilm formation was found to be dose dependent. Green-synthesized nanoparticles showed 99% inhibition against biofilm and bacteria, performed excellent anticancer assay with an IC50concentration of 17.1 ± 0.6 µg/mL and 100% inhibition, and photodegradation of the toxic organic dye Eosin Y up to 50%. Moreover, the effect of the pH and dosage of the photocatalyst was also measured to optimize the reaction conditions and maximum photocatalytic potential. Therefore, synthesized silver nanoparticles can be used in the treatment of wastewater contaminated with toxic dyes, pathogenic biofilms, and the treatment of cancer cell lines.