In the present investigation, synthesis and characterization of Zinc Oxide (ZnO) and Silver nanoparticles (NP), and their application on pathogenic bacteria were investigated. ZnO NP were synthesized by chemical reduction method using starch as capping agent and silver NP was prepared by green synthesis process from AgNO3 solution through the extract of Citrus sinensis (sweet lime). The detail characterization of the nanoparticles was carried out using UV-Vis spectroscopy, Dynamic Light Scattering (DLS) particle size analysis, Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD) analysis and Thermogravimatric (TGA) analysis. From Dynamic Light Scattering (DLS) particle size and SEM image analysis, the average particle size was found to be 90 nm and 50 nm, for ZnO and silver nanoparticles, respectively. From the analysis of XRD pattern, UV-VIS spectroscopy and TGA, the formation of nanoparticles was confirmed. Antibacterial assay of synthesized ZnO and silver NP was carried out both in liquid and solid growth medium against four pathogens (Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and streptococcus pneumonia). The bacterial growth was monitored by measuring the optical density (OD) of culture solution and estimation of colony forming units (CFU) on solid medium. The effect of NP on the level of gene expression in E.coli was also examined. When NP was administered in the liquid E.coli culture, considerable enhancement of the enzymatic activity of expressed β-glucosidase was observed. Further the physical interaction between bovine α-lactalbumin protein and NP was monitored by DLS particle size analyzer, tryptophan fluorescence and circular dichroism spectroscopy.