Background: Copper is a mineral that is involved in many functions og the human body and copper has antibacterial reaction. However many scientists have studied the antibacterial activity of nano copper, in our country, the lack of research is the basis of our research. Aim: Determination of antibacterial activity of colloidal copper (CuNP) Materials and Methods: The study protocol was reviewed and approved by the Research Ethics Review Committee of the Mongolian National University of Medical Sciences on February 18, 2022 (Protocol No. 2022/3-02). An ionized copper solution was produced by passing an electric current (19–20 V, 3–5 A, for 24 h) through sterilized distilled water using copper plates. Ascorbic acid was then added to prepare colloidal copper. The concentration was determined using a PPM meter, and the solution was filtered through a 0.22 µm bacterial filter. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against standard bacterial strains were determined by the broth dilution method. To determine the bactericidal time, bacterial cultures were exposed to the bactericidal concentration and subcultured hourly for 10 hours. Additionally, antibacterial activity was evaluated on Mueller–Hinton agar using the disk diffusion method according to CLSI standards, where 20 µL of colloidal copper was applied onto antibiotic disks. The plates were incubated at 37°C for 24 hours, and the diameter of the inhibition zones was measured. Result: The concentration of the prepared colloidal copper was 0.7 ppm. Using the broth dilution method, complete bactericidal activity was observed at 0.006 ppm against Klebsiella pneumoniae, Escherichia coli, and Salmonella enteritidis, while Pseudomonas aeruginosa was completely killed at 0.008 ppm. In terms of exposure time, complete bacterial killing occurred after 2 hours for S. enteritidis and K. pneumoniae, after 6 hours for E. coli, and after 11 hours for P. aeruginosa. In addition, antibacterial activity was evaluated on Mueller–Hinton agar using the disk diffusion method according to CLSI standards. Colloidal copper increased the inhibition zone diameter of most antibiotics by 1–5 mm, particularly against P. aeruginosa. In K. pneumoniae, the activity of aztreonam and ceftriaxone was enhanced. When evaluating the inhibition zone diameter of cefotaxime against E. coli, the susceptibility result changed from intermediate to susceptible, while no significant change was observed for the other antibiotics tested. For K. pneumoniae, the addition of colloidal copper increased the inhibition zone diameter of aztreonam and ceftriaxone by approximately 1–2 mm. In P. aeruginosa, the inhibition zone diameters of most antibiotics increased by 1–5 mm; however, the activity of imipenem decreased. For S. enteritidis, most antibiotics showed an increase in susceptibility of approximately 1–2 mm, while ceftriaxone showed no observable effect. Conclusion 1. Colloidal copper solution, at low concentrations, exhibits bactericidal activity against some Gram-negative rod-shaped bacteria. 2. Colloidal copper solution shows a synergistic effect with certain drugs, enhancing their activity; however, it exhibits an inhibitory effect on the activity of imipenem.