Green methods using biological extracts, in particular plant-based solutions, have shown great potential for silver nanoparticle synthesis. A microwave-assisted single-step phytosynthesis of silver nanoparticles is described in the present study. The aqueous extract obtained from the Rosa santana (rose) petals was used for the first time in the synthesis. The synthesized nanoparticles obtained after optimized microwave conditions for time and temperature were analyzed by ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and Zeta-size analysis. The results obtained from the characterization studies showed that the synthesized nanoparticles were nearly spherical in shape with sizes from 6.52 nm to 25.24 nm with an average particle size of 14.48 nm with a face-centered cubic structure. The antibacterial activities of the synthesized nanoparticles were evaluated and revealed that the silver nanoparticles displayed good inhibition against both Gram-negative and Gram-positive bacteria. Also, the cytotoxic effect of the silver nanoparticles on a mouse fibroblast cell line (L929) was studied by a cell viability assay. The results showed that phytosynthesized silver nanoparticles were nontoxic to the healthy normal cell line at all tested concentrations.