Nanofluids are liquids containing nanoparticles, nanotube or nanowire suspensions. This work investigates the disinfection properties of ZnO nanofluids, especially the change of ZnO disinfection properties when facing different shaking speeds of incubator (50rpm, 100rpm, 200rpm and 300rpm). In this work, E. coli was used as model bacteria to test the disinfection property of ZnO nanofluids. Plant counting method was used to examine the number of bacteria before and after the treatment of ZnO nanofluids. The results demonstrate that both ZnO nanofluids sample A and B could inhibit or kill E. coli bacteria. ZnO sample B with smaller particle size showed better antimicrobial activities. Treated with higher concentration of ZnO sample B exhibited better antimicrobial activities. Besides, this work also demonstrated that with higher shaking speed of incubator, ZnO nanofluids showed better antimicrobial activities. The antimicrobial mechanism is thought to be associated with physical interaction between ZnO particles and the biological cells and enhanced mass transfer of possible chemical species produced due to the presence of ZnO particles.