The antimicrobial activity of trimetallic Au/Pt/Ag nanoparticle based nanofluids were studied and compared with that of monometallic Au and bimetallic Au/Pt nanofluid. The trimetallic nanofluid was prepared by green microwave assisted successive chemical reduction method and characterized by UV-Vis spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM). The metallic nanofluids were tested for antibacterial properties using agar disc diffusion test method against various micro-organisms and their minimum inhibitory concentration (MIC) values were calculated. The trimetallic nanofluid exhibited efficient antibacterial activity and were found to be better agent than bimetallic and single metallic nanofluid at a very low metal concentration. These results of trimetallic nanofluids may be utilized in the field of many applications such as medical research, pharmaceutical industries and environmental sciences.