Silver nanoparticles biosynthesis has received increasing attention in the field of nanotechnology due to their antimicrobial and biomedical applications. Green synthesis of metal nanoparticles is anticipated as a cost effective and eco-friendly alternative in the current research scenario. With this aim, the aqueous extracts made from the aerial parts of Lippia nodiflora were used as the reducing agents to synthesize silver nanoparticles (AgNPs) and their antioxidant, antibacterial and cytotoxic properties have also been evaluated. The biosynthesized AgNPs were characterized by UV–Visible Spectroscopy, Fourier-Transform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD) analysis. The AgNPs were found to be stable at −25.2 mV through zeta potential study. The morphology and size of synthesized silver nanoparticles were confirmed by Scanning Electron Microscope with energy dispersive spectra (SEM-EDX) and Transmission Electron Microscopy (TEM) analysis with size range from 30 to 60 nm. Biosynthesized AgNPs exhibited strong antioxidant activity as well as showed potent antibacterial activity against human pathogenic bacteria. The cytotoxicity study of AgNPs was also revealed against MCF-7 breast cancer cell lines in a dose-dependent manner. The recognized bioactivity confirmed by the synthesized AgNPs directs towards the clinical use as an antioxidant, antibacterial and cytotoxic agent.