A simple, effective and environmentally friendly green synthesis was demonstrated to obtain trimetallic Au/Pt/Ag nanoparticles using Lamii albi flos extract. The formed nanoparticles (NPs) were characterized by Ultraviolet-Visible, Fourier-Transform Infrared, Scanning Electron Microscopy, Transmission Electron Microscopy, and Atomic Force Microscopy measurements. The applied physicochemical methods indicated that the size of synthesized Au/Pt/Ag NPs was in the range of 35-40 nm. The antimicrobial activity of the obtained nanoparticles was assessed using Gram-positive, Gram-negative bacteria and yeasts, including standard strains of Staphylococcus aureus NCTC 4163, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231. Also, antibacterial and antibiofilm activities of novel nanoparticles were studied using clinical strains of Enterococcus spp. The obtained Au/Pt/Ag NPs revealed excellent antimicrobial effects against planktonic and sessile clinical strains of Enterococcus faecalis and Enterococcus faecium.