Abstract This study explores a novel, eco-friendly approach to silver nanoparticle (AgNP) synthesis by using rose petal (RP) extracts. Unlike traditional chemical synthesis, this method reduces silver ions (Ag+) to elemental silver (Ag0) with RP, facilitating nanoparticle stabilization. RP-AgNPs were structurally characterized using UV/Vis (ultraviolet/visible) spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, zeta potential, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy which confirmed the synthesis of predominantly spherical to oval RP-AgNPs, with a distinct absorption peak at 410 nm as observed in UV/Vis spectra. The NPs demonstrated exceptional antibacterial efficacy against Escherichia coli and Staphylococcus aureus (inhibitory zones of 12.5 ± 0.20 and 14 ± 0.67 mm at 50 µg) and potent antibiofilm activity. Crucially, they also exhibited selective cytotoxicity toward HeLa cervical cancer cells when assessed by viability assays, fluorescence imaging, and flow cytometry. These findings underscore the potential of green synthesized AgNPs for future biomedical applications in areas such as infection control and cancer treatment.