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In this work, ZnO nanofluids were produced by a medium mill with a pH value of about 7.2 and characterized by Nano-Sizer and SEM. After milling, ZnO nanofluids were formed with an average particle size of～198.4 nm. The ZnO nanofluids used for testing were stored for different periods (1-, 90- and 120-day) and kept in different conditions (under the light and in the dark). The antibacterial activ- ities of these ZnO nanofluids were evaluated by estimating the reduction ratio of the bacteria treated with ZnO. The results showed that the ZnO nanofluid stored for 120 days under the light had the best antibacterial behavior against Escherichia coli DH5α. SEM images suggest that an interaction between the ZnO particles and the E. Coli bacteria cells caused by electrostatic forces might be a mechanism.