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The present study reports the simple and eco-friendly approach for biosynthesis of silver nanoparticles (AgNPs) using aqueous callus extract as reducing agent for the first time. The formation of AgNPs was initially confirmed by characteristic surface plasmon resonance (SPR) peak 453 nm by UV–Visible spectroscopy. FTIR spectrum shows different functional groups which probably involved in the synthesis and stabilization of AgNPs. TEM analysis determined the well-dispersed AgNPs with roughly spherical shape and size ranging 5–40 nm. XRD patterns revealed the crystalline nature of AgNPs with face-centered cubic (fcc) lattice. The synthesized AgNPs were found to have strong inhibitory activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa.