The in vitro callus induction of Solanum incanum L. was executed on MS medium supplemented with different concentrations of auxin and cytokinin utilizing petioles and explants of leaves. The highest significant fresh weights from petioles and leaf explants were 4.68 and 5.13 g/jar for the medium supplemented with1.0 mg L−1 BA and 1.0 mg L−1 2,4-D. The callus extract of the leaves was used for the green synthesis of silver nanoparticles (Ag-NPs). Analytical methods used for Ag-NPs characterization were UV-vis spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and Transmission Electron Microscopy (TEM). Spherical, crystallographic Ag-NPs with sizes ranging from 15 to 60nm were successfully formed. The FT-IR spectra exhibited the role of the metabolites involved in callus extract in reducing and capping Ag-NPs. The biological activities of Ag-NPs were dose-dependent. The MIC value for Staphylococcus aureus, Bacillus subtilis, and Escherichia coli was 12.5 µg mL−1, while it was 6.25 µg mL−1 for Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans. The highest inhibition of phytopathogenic fungi Alternaria alternata, Fusarium oxysporum, Aspergillus niger, and Pythium ultimum was 76.3 ± 3.7, 88.9 ± 4.1, 67.8 ± 2.1, and 76.4 ± 1.0%, respectively at 200 µg mL−1. Moreover, green synthesized Ag-NPs showed cytotoxic efficacy against cancerous cell lines HepG2, MCF-7 and normal Vero cell line with IC50 values of 21.76 ± 0.56, 50.19 ± 1.71, and 129.9 ± 0.94 µg mL−1, respectively.