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In the present study, antibacterial activity of silver nanoparticles synthesized from entophytic fungi extracts were analysed against biofilm forming bacteria include the Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa isolated from clinical specimens. Therefore, we tried to establish a combination of medicinal and nanotechnology possibly with the field of medicine for the development of antibacterial agents against these strains. The nanoparticles were characterized by UV–visible spectroscopy, the presented and capped molecules of proteins with nanoparticles was confirmed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD). UV–visible studies display an absorption band at 420 nm due to surface Plasmon resonance (SPR) of the silver nanoparticles. The intra cellular synthesized silver nanoparticles (Ag NPs) size 15-35 nm was determination of X-ray diffraction (XRD) crystal calculation, antimicrobial activity higher than the standard antibiotic. The nanoparticle treated with isolated bacteria as result macromolecule of proteins denatures and oozing out the cells and measure the Bradford method. The nanoparticle treated bacterial cells were compared to negative control more amount of protein released. The nano size establishing the silver nanoparticles directly bind with DNA of the pathogenic bacterial strains most important to higher antimicrobial activity.