Importance of hospital environment in patient-care has been recognized widely in infection prevention and control. Inappropriate antibiotic use led to emergence of resistant strains that are difficult to treat with the available antibiotics. Progress in nanotechnology led to enhancement of nanoparticles with physicochemical characteristics and functionality that overcomes the constraints of common antimicrobials. Aim was to investigate effective antimicrobial role of Silver nanoparticle (Ag-NPs) against clinically important bacterial strains and observe effects of varying storage temperatures on Ag-NPs antimicrobial activity. Different concentrations of Ag-NPs were tested against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii using diffusion method. Zone of inhibition (ZOI) for each organism was directly proportional to concentration of Ag-NPs used. Mean ZOI values at different concentrations were significantly different for all organisms with p-value <0.001 for E. coli, S. aureus, P. aeruginosa and 0.004 for A. baumannii. Variation in storage temperature hardly showed any effects on the antimicrobial property of the Ag-NPs. Scanning electron microscopy (SEM) showed morphological and size variations in Ag-NPs exposed cells when compared to control strains, especially for S. aureus, E. coli and P. aeruginosa. Damaged cell membrane areas can be clearly distinguished in E. coli and P. aeruginosa thus suggesting bacterial membrane disruption. These finding can help design a larger study where Ag-NPs can be used in various medical instruments which are usually kept at room temperatures. Also, outcomes of this study may help in designing proper implants, prosthesis and equipment coated with minimum concentration of nanoparticles that might be considered safe for medical applications.