Zinc oxide, as a typical semiconductor with a wide and direct-emission band gap, is a versatile material; it is also a biosafe and biocompatible material and can be used for biomedical applications without coating [1-3]. Well-define ZnO nanostructures with various morphologies such as nanowires and nanorods, nanobelts, helical nanorods and columns, nanotetrapods, nanotubes, nanosheets, and some complicated hierarchical nanostructures have been documented [4-6]. Due to the outbreak of the infectious diseases caused by different pathogenic bacteria, the researchers are searching for new antibacterial agents. In the present scenario, nanoscale materials have emerged up as novel antimicrobial agents owing to their high surface area to volume ratio and the unique chemical and physical properties [7]. In recent years, the use of inorganic antimicrobial agents has been attracted interest for the control of microbes. The key advantages of inorganic antimicrobial agents are improved safety and stability, as compared with organic antimicrobial agents [8]. At present, most antibacterial inorganic materials are metallic nanoparticles [9-11] and metal oxide nanoparticles such as zinc oxide [8]. In this work, a green and cost-effective method for the preparation of ZnO nanoparticles via microwave assisted decomposition of zinc acetate precursor using 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide, [bmim][NTf2] as a solvent has been used. The morphology and structure of ZnO nanoparticles have been characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM). ZnO nanofluids have been prepared by dispersing ZnO nanoparticles in glycerol as a base fluid in the presence of ammonium citrate as a dispersant. The antibacterial activity of ZnO nanofluids was tested against E. coli at different concentrations by colony count method. The quantitative examination of bacterial activity has been estimated by the survival ratio (N/N0) as calculated from the number of viable bacterial cells (N) at specified time and the initial viable cells (N0), which form colonies on the nutrient agar plates.