Background: Bearing in mind the global problem of rising number of infections caused by resistant microorganisms, it is crucial to develop new and better approaches in preventing and treating them. Despite the availability of numerous potent antibiotics and other antimicrobials, bacterial infections are still a major cause of morbidity and mortality. Therefore, exploring and developing new antimicrobial agents as antibiotic alternative is among key steps. As one of the most common human pathogens of potentially life-threatening infections, Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneumoniae) bacterial strains are among the most researched ones in this field of science. Aim: Since some metal and metal oxide nanoparticles have already shown antimicrobial effect, the aim of this study was to closely investigate the antimicrobial effect of colloidal platinum nanoparticles. Methods: The antimicrobial effect of nano platinum was tested on standard laboratory and clinical resistant E.coli and K.pneumoniae strains. Standard microbiological methods used to determine the antimicrobial effect of platinum nanoparticles were: microdilution method with determination of MIK and MBK values, agar well diffusion method and method for determining time-dependent antimicrobial effect or so-called “time-kill” assay. Results: Platinum nanoparticles expressed antibacterial effect among all tested bacterial strains. In addition, platinum nanoparticles showed to be bactericidal to all strains except K.pneumoniae ESBL+. Double-MIC value of colloidal platinum nanoparticles, which was previously determined for K.pneumoniae ESBL+ in the microdilution method, showed a bacteriostatic effect in the “time-kill” assay. Conclusion: Based on the results, in the group of metal and metal oxide nanoparticles with antimicrobial effect, platinum nanoparticles could be classified as potent ones. With additional research, not only in vitro but also under in vivo conditions, opportunities to exploit their antimicrobial potential in the future are opened.