The antibacterial activity of the biosynthesised Zinc Oxide nanofluid was measured against three gram positive species of bacteria and two gram negative species of bacteria. The gram positive species chosen were Enterococcus faecalis, Methicillin-resistant Staphylococcus aureus, and Staphylococcus saprophyticus, and the gram negative species were Escherichia coli and Pseudomonas aeruginosa. The zone of inhibition(ZOI) was measured for each of the bacteria after 24 hours of incubation at the temperature of 37°C. The performance of the ZnO nanofluid was the most effective against Staphylococcus saprophyticus and Pseudomonas aeruginosa, with a ZOI of 8mm, as illustrated in Table and Figure . The ZOI for Staphylococcus saprophyticus was uniform in a circular zone. The ZOI formed against Pseudomonas aeruginosa was not uniform and was rather randomly dispersed. The chosen positive antibiotic discs were Ciprofloxacin, Vancomycin, Meropenem. Cirpofloxacin being the same for all bacteria and Vancomycin for gram positive and Meropenem for gram negative. The performance and ZOI of the antibiotic discs for Staphylococcus saprophyticus and Pseudomonas aeruginosa was greater and more uniform than the ZnO nanofluid. The ZnO nanofluid also had a ZOI of 7mm for Enterococcus faecalis, with the zone spreading out in a crack-like form. The ZnO nanofluid had the lowest ZOI for Methicillin-resistant Staphylococcus aureus and Escherichia coli, coming in at 6mm. The ZOI for the Escherichia coli sample also had a crack-like pattern, however it was not as significant and uniform as Enterococcus faecalis. The comparison of antibacterial performance between ZnO nanofluid, Ciprofloxacin, Vancomycin, and Meropenem against the selected bacteria showed that the antibacterial activity of the ZnO nanofluid can be attributed to the production of reactive oxygen species (ROS) on the ZnO NP's surface and the release of Zn 2+ ions . These mechanisms cause damage to the bacterial cell walls, ultimately leading to the penetration of Zn 2+ ions . The inferior antibacterial performance of the ZnO NPs is likely due to their morphology. Given that the bacterial cells are considerably larger than the ZnO NPs , optimal interaction between the bacteria and the nanoparticles is not achieved. This suggests that the production of reactive oxygen species (ROS) on the ZnO NPs surface and the release of Zn 2+ ions are minimal. Consequently, the ZnO NPs exhibited lower antibacterial activity compared to Ciprofloxacin, Vancomycin, and Meropenem against all the tested bacteria.