With the advancement of nanobiotechnology, eco-friendly approaches of plant-mediated silver nanomaterial (AgNP) biosynthesis have become more attractive for biomedical applications. The present study is a report of biosynthesizing AgNPs using Chlorophytum borivilianum L. (Safed musli) callus extract as a novel source of reducing agent. AgNO3 solution challenged with the methanolic callus extract displayed a change in color from yellow to brown owing to the bioreduction reaction. Further, AgNPs were characterized by using UV-visible spectrophotometry, X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), and Fourier Transform Infrared Spectroscopy (FTIR). UV-vis spectrum revealed the surface plasmon resonance property of AgNPs at around 450 nm. XRD pattern with typical peaks indicated the face-centered cubic nature of silver. AFM analysis confirmed the existence of spherical-shaped and well-dispersed AgNPs having an average size of 52.0 nm. Further, FTIR analysis confirmed the involvement of different phytoconstituents of the callus extract role in the process of bioreduction to form nanoparticles. The AgNPs were more efficient in inhibiting the tested pathogenic microbes, namely, Pseudomonas aeruginosa, Bacillus subtilis, Methicillin-resistant Escherichia coli, Staphylococcus aureus, and Candida albicans compared to callus extract. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed the cytotoxic property of AgNPs against human colon adenocarcinoma cell line (HT-29) in a dose-dependent manner. At higher concentrations of 500 mu g/mL AgNPs, the cell viability was observed to be only 7% after 24 hours with IC50 value of 254 mu g/mL. Therefore, these AgNPs clearly endorse the manifold potential to be used in various biomedical applications in the near future.