Polyvinyl alcohol/chitosan/silver (PVA/CS/Ag) nanocomposite membranes were synthesized by gamma-radiation with promising antimicrobial and biomedical applications. The nanocomposite membranes were prepared by mixing PVA and CS solutions with different copolymer compositions in the presence of silver nitrate (AgNO3) and glutaraldehyde as cross-linker, followed by in situ reduction with gamma-radiation at different doses. The nanocomposite membranes were characterized by ultraviolet spectroscopy (UV), Fourier transform infrared, X-ray diffraction (XRD) and transmission electron microscopy (TEM). UV studies showed a strong peak around lambda (max) at 430 nm due to surface plasmon resonance of silver nanoparticles formed during irradiation. As the irradiation dose increased from 25 to 75 kGy, the plasmon band is shifted from 430 to 418 nm with high intensity, indicating the formation of smaller particles. TEM investigation showed uniform distribution of silver nanoparticles (AgNPs) in the membranes with mean diameter of 32-19 nm. XRD results confirmed that the mean diameter of AgNPs estimated from the Debye-Scherrer formula was in the range of 27.5-12.8 nm which confirms the TEM results. The PVA/CS/Ag nanocomposite membranes exhibited good antibacterial activity and were found to cause significant reduction in microbial growth. The nanocomposite membranes showed non-thrombogenicity effect and slightly haemolytic potential, suggesting their promising use in biomedical applications.