Background & Objectives: This study aimed to examine the antibacterial effect of Nano Polyamidoamine-G5 (NPAMAM-G5) dendrimer on Escherichia Coli, Proteus Mirabilis, Salmonella Typhi, Bacillus Subtilis and Staphylococcus Aureu s isolated from the hospital environment.Materials & Methods: In this study, the antibacterial effects of NPAMAM-G5 dendrimer were studied by disc diffusion and micro-dilution method. Different concentrations of Polyamidoamine-G5 inoculated onto blank disks and were placed in Mueller-Hinton agar media. Zone of inhibition was investigated by bacterial inoculation according to the McFarland standard 0.5. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of NPAMAM-G5 dendrimer were determined by micro-dilution method.Results: Zone of inhibition in Polyamidoamine-G5 dendrimers concentration of 25 mg/mL for Escherichia Coli, Proteus Mirabilis, Salmonella Typhi, Bacillus Subtilis and Staphylococcus Aureus were 22, 28, 21, 16 and 20 mm, respectively. Regarding the zone of inhibition in gram negative bacteria with gram positive ones was p=0.16 and was not significant different. The minimum inhibitory concentration for both Proteus Mirabilis and Salmonella Typhi was 2.5 mg/mL and for Escherichia Coli, Bacillus Subtilis and Staphylococcus Aureus was 25 mg/mL. The Minimum Bactericidal Concentration (MBC) for Salmonella Typhi was 50 mg/mL and for all Proteus Mirabilis, Escherichia Coli, Bacillus Subtilis and Staphylococcus Aureus was 100 mg/mL.Conclusion: According to the results, NPAMAM-G5 dendrimer can effectively eliminate Escherichia Coli, Proteus Mirabilis, Salmonella Typhi, Bacillus Subtilis and Staphylococcus Aureus.