As a consequence of growing water demand and pollution, waterborne disease outbreaks are on the rise, and current disinfection methods have shown to be ineffective in inactivating all pathogens during water treatment. Zinc oxide nanoparticles (ZnO NPs) have been reported to possess antimicrobial properties due to oxidative stress caused by reactive oxygen species. Also, ZnO has high thermal and chemical stability and low toxicity, which makes these NPs an excellent candidate for water treatment applications. Furthermore, incorporation of lithium in the crystal structure of ZnO NPs should enhance the production of reactive oxygen species. Accordingly, this work addresses the effect of Li doping on the bactericidal properties of ZnO nanorods synthesized using a polyol-based process. X-ray diffraction analyses confirmed the wurtzite ZnO phase; the incorporation of Li in the ZnO structure was evidenced by the systematic shift of the diffraction peaks. Scanning electron microscopy characterization was used to determine morphology and size. Ultraviolet–visible and photoluminescence spectroscopy measurements was used to evaluate the optical properties of the synthesized nanoparticles. The antibacterial activity of pure and doped ZnO NPs was evaluated against E. coli, S. typhimurium and E. faecalis. Minimum inhibitory concentration was determined using a simple microdilution method. Also, growth curves of the test bacterium were generated using the colony count method to better elucidate the possible bactericide mechanisms of synthesized nanoparticles.