Background: Biogenic silver nanoparticles (AgNPs) have wider range of biomedical applications. The present work synthesized Tp-AgNPs using mycelial extract of endophytic fungus Talaromyces purpureogenus (MEEF), characterized, and analyzed for antibacterial, anti-proliferation and cell wounding healing activities. Methods: The synthesized Tp-AgNPs were characterized by UV-visible spectrophotometer (UV-Vis), field emission transmission electron microscopy (FETEM) with energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), particle size analysis (PSA) and X-ray diffraction (XRD). Further, antibacterial activity was determined by Kirby–Bauer test and anti-proliferation activity was tested in human lung carcinoma A549 by water-soluble tetrazolium and flow cytometer assay. In addition, cell wounding healing activity was determined by scratch assay. Results: UV-Vis results displayed a strong absorption peak from 390 nm to 420 nm, which indicated the successful synthesis of Tp-AgNPs. FETEM-EDS results indicated the round and triangle shaped Tp-AgNPs with the average size of 25 nm in accordance with PSA. FTIR analysis indicated the involvement of various functional molecules from MEEF in the synthesis of Tp-AgNPs. XRD result proved nature of Tp-AgNPs as a high-quality crystal. The Tp-AgNPs significantly inhibited the growth of bacterial pathogens at the minimal inhibitory concentration of 16.12 μg.mL^−1, for Gram,⁺ and 13.98 μg.mL^−1, for Gram^−, bacteria. Further, Tp-AgNPs (2 μg.mL^−1,) showed a strong anti-proliferation effect in A549. Interestingly, Tp-AgNPs was not cytotoxic to normal NIH3T3 cells. In addition, the NPs exhibited a strong cell wounding healing activity. Conclusion: This work biosynthesized AgNPs with strong antibacterial, anticancer and cell wound healing properties using endophytic fungus T. purpureogenus.