Herein, we described for the first time, biosynthesis of silver nanoparticles (AgNPs) using pre-hydrolyzed liquor of Eucalyptus wood under ambient conditions. The pre-hydrolyzed liquor containing a high amount of metabolites such as polyphenols, hemicelluloses and its derivatives are mainly assisted for the reduction and stabilization process of Ag+ ions to AgNPs. The formation of AgNPs is monitored by recording the UV-vis spectrophotometer for surface plasmon resonance (SPR) peak observed at ∼415nm. The intensity of SPR increased linearly with increasing the reaction time at ambient condition. X-ray diffraction (XRD) pattern of AgNPs reveals the formation of face-centered cubic structure. Field emission electron microscopy (FESEM) and transmission electron microscopy (TEM) images show the spherical shaped particles and narrow size distribution with an average diameter of 25-30nm. The elemental analysis by energy dispersive X-ray (EDAX) analysis confirms the presence of Ag as the major amount and is found to be 82%. Analysis of the fourier transform infrared (FT-IR) spectra of the NPs revealed the presence of phytoconstituents from pre-hydrolyzed liquor adsorbed on the surface of AgNPs. Moreover, in vitro, antimicrobial activity is found to be effective for as-synthesized AgNPs on tested bacteria (viz., P. aeruginosa, S. aureus and E. coli) followed by fungus (C. oxysporum, P. chrysogenum, C. albicans and A.niger). Thus, these results suggest the use of biosynthesized AgNPs as effective growth inhibitors for various biomedical applications.