<p>The green synthesis is an efficient, cost effective and eco-friendly method used for the production of nanomaterials. In the present study, callus extract of <i>Salix tetrasperma</i> was used for the first time for the synthesis of silver nanoparticles (Ag-NPs). The production of Ag-NPs was initially observed by the colour change in reaction mixture, which was subsequently confirmed by ultraviolet–visible absorption (UV–Vis) spectroscopy, showing a surface resonance peak between 400 and 450 nm. Crystallographic features were verified by X-ray diffraction (XRD), which showed characteristic diffraction peaks at 2θ values corresponding to the (111), (200), (220), and (311) planes of face-centered cubic silver. Fourier transform infrared (FTIR) spectroscopy identified functional groups present in the extract, and energy-dispersive X-ray spectroscopy (EDX) determined the elemental composition. The scanning electron microscopy (SEM) of Ag-NPs showed distinct and irregular morphology whereas transmission electron microscopy (TEM) revealed their spherical shape with an average particle size ranging from 6 to 30 nm. The antimicrobial potential of the biosynthesized Ag-NPs was assessed, showing a 66.82% inhibition rate against the phytopathogenic fungus <i>Macrophomina phaseolina</i> at 100 mgL^-1. The highest hatching inhibition and mortality rates for the root-knot nematode <i>Meloidogyne incognita</i> were 95.54% and 93.32%, respectively, at 100 mgL⁻¹ after 120 hours of treatment. The Ag-NPs exhibited significant antibacterial activity against <i>Pseudomonas aeruginosa</i> PAO1 at a minimal inhibitory concentration (MIC) value of 20 μg/mL. Additionally, dose-dependent anti-biofilm activity was observed, with a significant reduction at concentrations of 10 μg/mL (MIC/2) and 5 μg/mL (MIC/4). The molecular docking analyses revealed the possible interaction between silver and biofilm-associated proteins, indicating specific binding to particular amino acid residues.</p>