The goal of the present study is on an eco-friendly approach for the biosynthesis of silver nanoparticles (AgNPs) using the aqueous tuber extract of the medicinal plant Gloriosa superba Linn. The physicochemical characteristics of Gloriosa superba tuber extract derived silver nanoparticles (GST-AgNPs) were confirmed using different analytical techniques. The GST-AgNPs resulted a spherical morphology with a size of about 7 nm–14 nm and the average size of ∼12 nm with no impurities and highly stable particles. FTIR study revealed that the CN group acts as a reducing agent as it strongly attracts the electron and bioreduced the silver ions from the tuber extract. The biological synthesized GST-AgNPs (40 μg/mL) exhibited potential antibacterial activity on Enterococcus faecalis (29 mm), followed by Bacillus subtilis (24 mm) and Staphylococcus aureus (23 mm), respectively. On the other hand, GST-AgNPs showed potent anticancer activity against human lung cancer cell line (A549) by decreasing cell proliferation and inducing apoptosis at a concentration of 46.54 μg/mL. Therefore the present study suggests that the biologically synthesized GST-AgNPs can be exploited more for potential biomedical applications.